Microscope imaging apparatus and biological-specimen examination system

ABSTRACT

A microscope imaging apparatus and a biological-specimen examination system that can accurately carry out measurement even for an object under examination having substantial brightness non-uniformity are provided. The microscope imaging apparatus includes a stage that holds the object under examination, an illumination unit that illuminates the object under examination, an image-acquisition unit that acquires images of the object under examination, and a motion unit that moves the stage and the image-acquisition unit relative to each other. The image-acquisition unit includes an imaging device capable of image acquisition using a time delay integration method. When acquiring a plurality of images of the object under examination, the exposure time during which accumulated charge is produced in the imaging device is made different for each of the acquired images, and the plurality of images are combined into a single image.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a microscope imaging apparatus and to abiological-specimen examination system using the same.

2. Description of Related Art

A known apparatus for measuring fluorescence and so on of a biologicalspecimen uses a microplate. Since the fluorescence intensity of abiological specimen is extremely dark, it is necessary to capture thefluorescence using a long exposure time. Also, since the microplate hasa shape that is larger than the field of view of a microscope, it takessome time to examine the entire microplate.

One method of imaging such fluorescence is a method in which measurementis carried out with a CCD (charge coupled device) while the microplateis repeatedly moved and stopped. A plurality of wells are provided inthe microplate, and the specimens are held in these wells. Therefore, byrepeatedly moving and stopping the microplate, the fluorescence from thespecimen in each well can be measured. The microplate is moved by meansof a moving stage.

Another known method of imaging the fluorescence is a method using TDI(time delay integration) imaging devices (for example the pamphlet ofInternational Publication No. 03/014400 (FIG. 1, etc.)). The imagingdevices using the TDI method are constituted by a plurality ofoptoelectronic devices. Fluorescence from the specimen is incident onthe optoelectronic devices, and a charge corresponding to the incidentfluorescence is produced in the optoelectronic devices byoptical-to-electrical conversion. This charge is then transferredbetween optoelectronic devices as the microplate moves. Since the motionof the microplate is associated with the motion of the charge,fluorescence from the same position on the specimen is incident again onthe optoelectronic devices after they have been moved. As a result, thecharge builds up. Thus, the charge is progressively accumulated whenusing the TDI method.

The feature of the TDI method is that charge corresponding to thefluorescence is accumulated while being transferred. Therefore, comparedto the case where a one-dimensional line sensor is used for imageacquisition, the speed at which the stage is moved can be increasedaccording to the number of charge transfer lines. As a result, themeasurement time can be shortened.

BRIEF SUMMARY OF THE INVENTION

A microscope imaging apparatus according to the present inventioncomprises:

-   -   a stage that holds an object under examination;    -   an illumination unit that illuminates the object under        examination;    -   an image-acquisition unit that acquires images of the object        under examination;    -   a motion unit that moves the stage and the image-acquisition        unit relative to each other; and    -   a control unit that controls the image-acquisition unit and the        motion unit,    -   wherein the image-acquisition unit includes an imaging device        that is capable of acquiring images with the time delay        integration method, and    -   when image acquisition of the object under examination is        carried out a plurality of times, the control unit makes the        exposure times of the imaging device different for each image        acquisition.

Another microscope imaging apparatus according to the present inventioncomprises:

-   -   a stage that holds an object under examination;    -   an illumination unit that illuminates the object under        examination;    -   an image-acquisition unit that acquires images of the object        under examination;    -   a motion unit that moves the stage and the image-acquisition        unit relative to each other; and    -   a control unit that controls the image-acquisition unit and the        motion unit,    -   wherein the image-acquisition unit includes an imaging device        that is capable of acquiring images with a time delay        integration method, and    -   by performing a prescan of the object under examination to        obtain a detected intensity of the object under examination, the        control unit determines an exposure time for image acquisition        after the prescan on the basis of the detected intensity.

Another microscope imaging apparatus according to the present inventioncomprises:

-   -   a stage that holds an object under examination;    -   an illumination unit that illuminates the object under        examination;    -   an image-acquisition unit that acquires images of the object        under examination;    -   a motion unit that moves the stage and the image-acquisition        unit relative to each other; and    -   a control unit that controls the image-acquisition unit and the        motion unit,    -   wherein the image-acquisition unit includes an imaging device        that is capable of image acquisition using two methods;    -   the control unit includes:        -   an examination-object-parameter input unit for inputting            information about the object under examination as an            examination-object parameter;        -   a calculation unit that calculates a time for the relative            motion on the basis of the examination-object parameter            which has been input; and        -   a switching unit that switches the image-acquisition method            of the imaging device on the basis of the calculation            result; and    -   the two image-acquisition methods are a time delay integration        method and a two-dimensional imaging method in which accumulated        charge is produced by a single exposure.

A biological-specimen examination system according to the presentinvention comprises:

-   -   a culture unit for culturing a biological specimen; and    -   a detection unit disposed adjacent to the culture unit,    -   wherein the detection unit includes:        -   an above-described microscope imaging apparatus; and        -   a preserving unit for preserving the biological specimen in            a predetermined state.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIGS. 1A to 1C depict an image acquisition operation using the TDImethod.

FIGS. 2A to 2C depict the transfer of signal charge in the TDI method.

FIGS. 3A to 3C show the intensity of signal charge accumulated inhorizontal lines.

FIG. 4 shows the overall configuration of a microscope imagingapparatus.

FIGS. 5A and 5B show the structure of a specimen.

FIG. 6 depicts scanning in the TDI method.

FIG. 7 is a flowchart showing a measurement procedure according to afirst embodiment.

FIGS. 8A to 8C are graphs showing the relationship between specimenbrightness and output level.

FIG. 9 is a timing chart for an electronic shutter used in the TDImethod.

FIG. 10 shows another example of the structure of a specimen.

FIG. 11 shows the overall configuration of a microscope imagingapparatus according to a second embodiment.

FIG. 12 is a flowchart showing a measurement procedure in the secondembodiment.

FIG. 13 is a graph showing the relationship between the number ofsaturated pixels and the exposure time used in the next imageacquisition.

FIG. 14 shows the overall configuration of a microscope imagingapparatus according to a third embodiment.

FIG. 15 depicts an image acquired in the third embodiment.

FIG. 16 is a flowchart showing a measurement procedure in a fourthembodiment.

FIG. 17 is a graph showing the relationship between prescan brightnessand exposure time of the main measurement.

FIG. 18 shows another example of the microscope imaging apparatus in thefourth embodiment.

FIG. 19 shows the overall configuration of a microscope imagingapparatus in a fifth embodiment.

FIG. 20 shows the overall configuration of a microscope imagingapparatus in a sixth embodiment.

FIG. 21 shows the overall configuration of a microscope imagingapparatus in a seventh embodiment.

FIG. 22 is a flowchart showing a measurement procedure in the seventhembodiment.

FIGS. 23A and 23B depict the structure of a specimen in an eighthembodiment.

FIG. 24 shows the overall configuration of a microscope imagingapparatus in a ninth embodiment.

FIGS. 25A and 25B show parameters used in estimating the scanning time.

FIG. 26 depicts scanning in a two-dimensional imaging method.

FIG. 27 depicts the operation of a stage in the two-dimensional imagingmethod.

FIG. 28 shows a specimen in which the density of sample located portionsis asymmetric.

FIG. 29 depicts an example in which scanning is switched between the TDImethod and the two-dimensional imaging method.

FIG. 30 is a perspective view showing a biological-specimen examinationsystem according to a tenth embodiment of the present invention, whichis provided with a microscope imaging apparatus.

FIG. 31 is a schematic diagram showing the system configuration of thebiological-specimen examination system shown in FIG. 30.

FIG. 32 is a perspective view of an incubator box used in thebiological-specimen examination system shown in FIG. 30.

FIG. 33 is a cross-sectional view of a chamber in the incubator boxshown in FIG. 32.

FIGS. 34A and 34B are perspective views showing other examples of theincubator box shown in FIG. 32.

FIGS. 35A to 35D show examples of selecting the scanning method and thedetection regions in the biological-specimen examination system.

FIG. 36 is a flowchart showing a procedure for setting measurementparameters used in the biological-specimen examination system.

FIGS. 37 and 38 are flowcharts showing a measurement procedure in thebiological-specimen examination system.

FIG. 39 is a flowchart showing an image processing method in thebiological-specimen examination system.

FIG. 40 is a flowchart showing a data processing procedure in thebiological-specimen examination system.

FIG. 41 is a flowchart showing a procedure for adjusting intensity inthe biological-specimen examination system.

FIG. 42 is a flowchart showing the method of supplying and replacingculture fluid in the biological-specimen examination system.

FIG. 43 shows a cell-tracking image representing the motion of cellsover time.

FIGS. 44 and 45 are flowcharts showing culturing and measurement using amicroplate.

FIGS. 46A and 46B are, respectively, an elevational view and a side viewof a biological-specimen examination system according to an eleventhembodiment, in which a microscope imaging apparatus is provided.

FIG. 47 is a plane view of a culture stage in the biological-specimenexamination system shown in FIGS. 46A and 46B.

FIG. 48 is a perspective view of the culture stage in thebiological-specimen examination system shown in FIGS. 46A and 46B.

DETAILED DESCRIPTION OF THE INVENTION

A microscope imaging apparatus will be described below with reference toFIGS. 1A to 6.

First, an image-acquisition operation according to a time delayintegration (TDI) method will be described using FIGS. 1A to 1C.

FIGS. 1A, 1B, and 1C depict the TDI image-capturing operation. FIGS. 2A,2B, and 2C depict the transfer of signal charge in the TDI method. FIG.3A shows the level of signal charge accumulated in a horizontal line LA,FIG. 3B shows the level of signal charge accumulated in a horizontalline LB, and FIG. 3C shows the level of signal charge accumulated in ahorizontal line LC.

For the sake of simplifying the explanation, a star-shaped specimen isassumed here. However, the shape of the specimen is not particularlylimited to this shape.

The positional relationship between a specimen (subject underexamination) 20 and an imaging area S of an imaging device 63 is shownin FIG. 1A. Image-acquisition begins when the top of the specimen 20 andthe bottom of the imaging area A overlap each other. Thus, initially,only the tip of the upward-facing point of the star projects onto theimaging device 63. At this time, as shown in FIG. 2A, the image of thetip of the upward-facing point is acquired by the horizontal line LA ofthe imaging device 63. As shown in FIG. 3A, signal charge with a levelcorresponding to the light intensity (brightness) from the specimen 20is accumulated in the horizontal line LA.

Subsequently, a stage 30 is moved with a predetermined timing. As shownin FIG. 1B, the stage 30 is moved in the positive Y direction by adistance corresponding to one horizontal line of the imaging device 63.By doing so, the entire upward-facing point of the specimen 20 isimaged.

At this point, as shown in FIG. 2B, the signal charge accumulated in thehorizontal line LA is transferred to the horizontal line LB insynchronization with the motion of the stage 30. Thus, the image of thespecimen 20 is acquired by the horizontal lines LA and LB of the imagingdevice 63.

Therefore, as shown in FIG. 3B, the signal charge accumulated duringthis image acquisition is added to the signal charge accumulated in theprevious acquisition. In other words, the signal charges obtained due tothe previous and current image acquisition are accumulated in thehorizontal line LB. As a result, the signal charge level is twice ashigh as that measured in one line.

In this way, charge transfer is carried out from the state shown in FIG.2A to the state shown in FIG. 2B. The time interval at which this chargetransfer is carried out is called the TDI line transfer rate.

The stage 30 is then moved further at the predetermined timing. That is,as shown in FIG. 1C, the stage 30 is again moved in the positive Ydirection by an amount corresponding to one horizontal line of theimaging device 63. By doing so, image-acquisition of the specimen 20proceeds.

At this point, as shown in FIG. 2C, the signal charge accumulated in thehorizontal line LB is transferred to the horizontal line LC insynchronization with the motion of the stage 30. In addition, the signalcharge accumulated in the horizontal line LA is transferred to thehorizontal line LB. Thus, the image of the specimen 20 is acquired bythe horizontal lines LA, LB, and LC.

Therefore, as shown in FIG. 3C, the signal charge obtained during thisimage-acquisition is added to the horizontal line LC. Thus, the signalcharges obtained by the previous-but-one image-acquisition, the previousimage-acquisition, and the current image-acquisition are accumulated inthe horizontal line LC. In other words, the level of signal charge isthree times higher than that measured with a single line. The signalcharge obtained in the previous image-acquisition and the currentimage-acquisition are accumulated in the horizontal line LB.

By continuing with the above-described operation, the same number ofimage-acquisition operations as the number of horizontal scanning linesis performed. Accordingly, a signal charge for the same part of thespecimen 20 is accumulated corresponding to the number ofimage-acquisition operations. Thus, with the TDI method, the image ofthe specimen 20 projected onto the imaging surface is shifted along withthe motion of the stage 30, and the signal charge accumulated in theimaging device 63 is shifted in synchronization therewith.

Next, the exposure time used when carrying out image-acquisition in theTDI method will be discussed.

The exposure time in the TDI method is the time it takes for the signallight returning from the specimen 20 to be accumulated as the signalcharge. In other words, the exposure time in the TDI method can beexpressed as the product of the TDI line transfer rate and thecumulative number of pixels. Therefore, to change the exposure time, theTDI line transfer rate can be made faster to lengthen the exposure timeor the TDI line transfer rate can be slowed down to shorten the exposuretime.

For example, when using a CCD camera having 1,000 pixels in the Ydirection as the imaging device 63, to acquire images with an exposuretime of 0.2 s, the line transfer rate is set to 5 kHz (0.2 ms transfertime) according to the calculation shown below:0.2 s/1000=0/2 ms.

Next, the stage scanning speed used in the TDI method will be discussed.

The stage 30 is moved in synchronization with the transfer of the signalcharge. Therefore, the speed at which the stage 30 is moved can beexpressed as the pixel size on the stage 30 divided by the TDI linetransfer rate. The pixel size on the stage 30 can be obtained from thepixel size of the imaging device 63 (for example, a CCD) and theprojection magnification.

For example, with a projection magnification of 10, a pixel size in theimaging device 63 of 6.45 μm, and a TDI line transfer rate of 5 kHz, thespeed of the stage is 3.23 mm/s, according to the calculation shownbelow:6.45×10⁻³ mm/10×5×10³ Hz=3.23 mm/s.

Next, the structure of a microscope imaging apparatus will be described.

FIG. 4 shows the overall configuration of a microscope imaging apparatus10. The microscope imaging apparatus 10 shown in FIG. 4 is used in thefirst to third embodiments of the present invention described below.

As shown in FIG. 4, the microscope imaging apparatus 10 includes thestage 30, an illumination unit 40, and an image-acquisition unit 60. Thestage 30 holds the specimen (object under examination) 20 thereon and ismoveable. The illumination unit 40 radiates illumination light onto thespecimen 20. The image-acquisition unit 60 acquires signal light emittedfrom the region irradiated with the illumination light and measures it.

The illumination unit 40 includes components capable of Kohlerillumination. Specifically, these are a lamp 41, a collector lens 42, areflecting mirror 43, a field stop 44, and a lens 45. A gas dischargelamp or the like, such as a halogen lamp, xenon lamp, or mercury lamp,is used at the light source lamp 41. The collector lens 42 collectslight emitted from the lamp 41. The reflecting mirror 43 reflects lighttraveling backwards from the lamp 41 back towards the lamp 41 again. Thefield stop 44 is disposed after the collector lens 42. This field stop44 is disposed at a conjugate position with respect to the focal pointof an objective lens 49 described below. The diameter of the opening ofthis field stop 44 is adjustable. A lens 45 is disposed after the fieldstop 44. The lens 45 images the field stop 44 at infinity.

An aperture stop 46 is disposed after the lens 45, at the focal planethereof. The diameter of the opening of this aperture stop 45 can bevaried. By doing so, the size of the beam diameter at the exit-pupilposition of the objective lens 49 can be adjusted. A mirror 47 isdisposed after the aperture stop 46. A dichroic mirror that reflectslight from the lamp 41 and that transmits return light (fluorescence)from the specimen 20 is used as the mirror 47. A half-mirror may be usedinstead of a dichroic mirror.

The objective lens 49 is disposed after the mirror 47. The stage 30 isdisposed at a position facing the objective lens 49.

A stage driving mechanism (motion unit) 31 for driving the stage 30 isprovided. The stage driving mechanism (motion unit) 31 drives the stage30 in the X and Y directions on the basis of a signal output from acomputer described below. Known technology, for example, a slidingmotion mechanism, may be used as the stage driving mechanism 31. It isnot particularly limited to a sliding motion mechanism, however.

An imaging lens 61 and a detector 62 are provided in theimage-acquisition unit 60. Return light (fluorescence) from the specimen20 is incident on the imaging lens 61, and the imaging lens 61 focuses(images) the incident light at a predetermined position. The detector 62is disposed at a predetermined position and detects the return lightfrom the specimen 20. The imaging device 63, which can acquire images bythe TDI method, is provided in the detector 62. As described above, theimaging device 63 accumulates signal charge in each horizontal line inresponse to the movement of the stage 30.

The output of the detector 62 is connected to an image processing unit64, which processes the output signal from the detector 62. A monitor 65for displaying the processed signal is connected to the image processingunit 64. Also, the image processing unit 64 is connected to the computer66. The stage driving mechanism 31 is also connected to the computer 66.

The computer (calculating unit) 66 calculates the exposure time in themain measurement. This calculation is carried out on the basis ofinformation obtained during a prescan, as described above. Thisinformation is the brightness in each sample located portion (describedbelow).

FIG. 5A depicts the shape of the specimen 20. FIG. 5B is a magnifiedview showing a sample located portion 22 of the specimen 20.

As shown in FIG. 5A, the specimen 20 includes a transparent substrate21. This transparent substrate 21 is formed of, for example, a glassplate or a plastic plate. The sample located portions 22 are formed in amatrix on the transparent substrate 21. That is, a two-dimensionalpatterned portion 23 is formed by the plurality of sample locatedportions 22.

The sample located portions 22 have a substantially circular shape witha diameter of a few millimeters in plane view and have a concave profilein cross-section. As shown in FIG. 5B, cells, serving as the objects tobe measured, are disposed in the sample located portions 22. The cellsare cultured inside the sample located portions 22.

Next, stage scanning in the TDI method will be described.

FIG. 6 depicts the scanning in the TDI method. In this embodiment, thestage 30 is moved to perform scanning. However, in FIG. 6, for the sakeof simplifying the drawing, the imaging device 63 is depicted as moving.

As shown in FIG. 6, when measuring the specimen 20 with the TDI method,the two-dimensional patterned portion 23 is completely scanned. Thisscanning is performed regardless of whether or not the sample locatedportions 22 are present.

When the stage 30 is scanned in the Y-axis direction, the stage 30 movesat constant velocity in the −Y direction. On the other hand, the imagingdevice 63 is capable of transferring charge only in one direction.Therefore, as shown in FIG. 6, measurement is carried out only while thestage 30 is advancing in the −Y direction. Measurement is not carriedout while the stage 30 is advancing in the +Y direction. The motion inthe +Y direction is for returning the stage to its initial position. Thetime required to return to the initial position may correspond to, forexample, the time required for storing the acquired data in a hard diskof the computer 66.

First Embodiment

Next, a first embodiment of the present invention will be described.Here, a measurement procedure will be described using FIG. 7.

FIG. 7 is a flowchart showing the measurement procedure in thisembodiment. FIGS. 8A to 8C show the relationship between the brightnessof the specimen 20 and the output level from the imaging device 63. FIG.8A shows the relationship between the brightness obtained in a firstscan and the output level, FIG. 8B shows the relationship between thebrightness obtained in a second scan and the output level, and FIG. 8Cshows the dependency of the brightness obtained in the first and secondscans no the output level.

First, the first scan (image acquisition) is performed to acquire animage of the specimen 20 (step S1). This corresponds to the prescan.

In this step, while the specimen 20 (stage 30) is moved from position ato position b shown in FIG. 6, the specimen 20 is imaged with apredetermined exposure time.

The image acquired in the first scan will be described in terms of therelationship between the brightness of the image and the output levelfrom the imaging device 63. In this case, as shown in FIG. 8A, there isa region where the accumulated charge is saturated (hereinafter referredto as saturation-level region) and a region where the accumulated chargeis low (hereinafter referred to as low-level region).

Next, preparation for the second scan is carried out (step S2).

The output levels of the saturation-level region and the low-levelregion are appropriately set. More specifically, exposure times ofsuitable length are set for each of these regions.

That is, when the saturation-level region is to be eliminated, theexposure time is set to be shorter than the exposure time in the firstscan. At this time, the output level is set to utilize the entiredynamic range of the imaging device 63. When the low-level region is tobe eliminated, the exposure time is set to be longer than the exposuretime in the first scan. In this case too, the output level is set toutilize the entire dynamic range of the imaging device 63.

Simultaneously, the specimen 20 (stage 30) is moved from position b toposition a.

This embodiment is described in terms of an example in which thesaturation-level region is eliminated.

Next, a second scan (image acquisition) is performed to acquire an imageof the specimen 20 again (step S3).

In this image acquisition, the exposure time determined in step S2 isused to acquire an image of the specimen 20 in the same way as in stepS1.

The image acquired in the second scan is a graph indicating therelationship between the brightness of the acquired image and the outputlevel of the imaging device 63. The image acquired in the second scan isshown in FIG. 8B. As shown in this figure, the saturation in thesaturation-level region in the previous scan is eliminated, whereas theoutput level is increased in the low-level region.

Next, the images acquired in the first and second scans are combinedinto a single image (step S4). A predetermined image processingoperation is carried out during this image combining.

In this step, the images acquired in steps S1 and S3 are combined. Bydoing so, a single image with wider dynamic range can be obtained.

Thus, in this embodiment, as shown in FIG. 8C, the image acquired instep S1 with the long exposure time and the image acquired in step S3with the short exposure time are combined to form a single image.

Thereafter, the specimen 20 (stage 30) is moved from position b toposition c, and the process proceeds to preparation for measuring thenext line.

This embodiment has been described in terms of an example in whichscanning of the same position is repeated twice. However, the number ofscans is not limited to two. For example, scanning may be repeated atthe same position three or more times. In such a case, between the imageacquisitions performed in each scan, preparation for the next scan iscarried out, and the acquired images are combined at the end.

Instead of the imaging device 63 described above, an imaging device 63 ahaving an electronic shutter may be used. Use of such an imaging device63 a is preferable since it can reduce the exposure time.

The electronic shutter is the type of shutter normally used in CCDdigital cameras and mobile telephones with built-in cameras. Such anelectronic shutter uses a phenomenon whereby charge is not accumulatedif the CCD is not operating, even if exposed to light. Accordingly, theCCD function itself is one form of shuttering.

On the other hand, in a mechanical shutter used in a silver-halidecamera, an opaque plate is placed between the lens and the film, andexposure is performed by opening and closing (moving) this plate. Thus,the electronic shutter is different from the mechanical shutter. Theelectronic shutter controls the exposure time without using a mechanicalshutter.

FIG. 9 is a timing chart for the electronic shutter in the TDI method.

As shown in FIG. 9, for example, when the maximum transfer rate in theTDI method is t1 (seconds), the charge is transferred to a neighboringhorizontal line every t1 seconds. The timing of the transfers isindicated by the arrows in the figure.

The electronic shutter is open for a period of t2 seconds from eachcharge transfer, and charge accumulation is performed. In other words,the charge accumulation of the CCD is carried out for t2 seconds afterevery charge transfer. On the other hand, the exposure time when notusing the electronic shutter is t1. Therefore, the ratio of the exposuretime when using the electronic shutter to the exposure time when notusing the electronic shutter is t2/t1.

For example, assume that the imaging device 63 a has 1000 pixels in theY direction and a maximum transfer rate (t1) of 10 kHZ (0.1 ms). Whenusing this imaging device 63 a to acquire images with an exposure timeof 1 ms, the open time of the electronic shutter (t2) should be set to0.001 ms, according to the calculation shown below:1000×0.1 (ms)×(t2/0.1)=1 (ms)t2=0.001 ms

When the electronic shutter is not used, the shortest exposure time isdetermined by the product of the maximum TDI transfer rate and thecumulative number of pixels. Accordingly, by using an electronic shutterof the type described above, it is possible to increase the range ofexposure times for which measurement is possible. Therefore, even ahigh-brightness specimen 20 can be accurately measured.

With the configuration described above, two images obtained by two scanswith different exposure times in steps S1 and S3 are combined into asingle image (S4). By doing so, it is possible to acquire an image witha wider dynamic range compared to an image acquired with a singleexposure time. Therefore, it is possible to accurately carry outmeasurement even for a specimen 20 whose brightness variation is large.

As shown in FIGS. 5A and 5B, the specimen 20 may include only the samplelocated portions 22 on the transparent substrate 21. Alternatively, asshown in FIG. 10, a reference line BL may also be formed in a regionclose to the left-hand side of the transparent substrate 21.

If the reference line BL is formed, it can serve as a reference whencombining the images acquired in each scan. Accordingly, it is possibleto simplify the image combining operation.

In this embodiment, since fluorescence examination is carried out, it ispreferable that the reference line BL be formed of a material thatfluoresces.

Second Embodiment

Next, a second embodiment of the present invention will be describedwith reference to FIGS. 11 to 13.

The basic structure of the microscope imaging apparatus of thisembodiment is the same as that of the first embodiment, but the methodof measuring the specimen is different from that in the firstembodiment. Therefore, in this embodiment, only the method of measuringthe specimen shall be described, using FIGS. 11 to 13, and a descriptionof the TDI method and so on shall be omitted.

FIG. 11 depicts the overall configuration of a microscope imagingapparatus 110 of this embodiment.

As shown in FIG. 11, the microscope imaging apparatus 110 includes astage 30, an illumination unit 40, and an image-acquisition unit 160.The stage 30 holds a specimen 20 and is moveable. The illumination unit40 irradiates the specimen 20 with illumination light. Theimage-acquisition unit 160 acquires signal light emitted from the regionirradiated with illumination and measures it.

An imaging lens 61 and a detector 62 are provided in theimage-acquisition unit 160. Return light from the specimen 20 isincident on the imaging lens 61, and the detector 62 detects the returnlight.

The detector 62 is connected to an image processing unit 64, whichprocesses the output of the detector 62. The image processing unit 64, amonitor 65 for displaying the processed signal, and a stage drivingmechanism 31 are connected to a computer 166.

An exposure-time input unit 167 and a setting unit 168 are provided inthe computer 166. A maximum exposure time Tmax is input using theexposure-time input unit 167. This maximum exposure time Tmax is relatedto measurement of the specimen 20 and is set by the user. The settingunit 168 is used to set an exposure time Tmeasure used for imageacquisition and measurement.

Next, the measurement procedure will be described.

FIG. 12 is a flowchart showing the measurement procedure in thisembodiment.

First, the user sets the maximum exposure time Tmax (step S10) byinputting the maximum exposure time Tmax to the exposure-time input unit167.

Next, a first scan (image acquisition) is performed to acquire an imageof the specimen 20.

In this step, the maximum exposure time Tmax input to the setting unit168 is set as the exposure time Tmeasure used for image acquisition andmeasurement. Therefore, an image of the specimen 20 is acquired with themaximum exposure time Tmax.

Next, preparation is arried out for a second scan (step S12).

In this step, the setting unit 168 sets the exposure time Tmeasure forthe second scan on the basis of the magnitude (number of saturatedpixels) in the saturation-level region.

More specifically, as shown in FIG. 13, the reltionship (table) betweenthe number of saturated pixels and the exposure time in the next imageacquisition is stored in the setting unit 168. On the basis of thistable, the computer 66 sets the exposure time Tmeasure for the secondscan. Thus, as the number of saturated pixels, for example, in eachsample located portion 22 increases, the exposure time for the secondscan decreases.

Saturated pixels shall now be explained. For example, if a 12-bit CCD isused as the imaging device 63, the maximum number of gradation levels is4095 (=2¹², the maximum value that can be represented by 12 bits).Therefore, a saturated pixel is a pixel for which the imaging deviceoutputs a value of 4095, in other words, a pixel whose output valuereaches the upper limit.

Next, a second scan (image acquisition) is performed to acquire an imageof the specimen 20 (step S13), and the images acquired in the first andsecond scans are combined to form a single image (step S14).

The measurement procedure after step S13 is the same as in the firstembodiment, and therefore it is merely shown in FIG. 12, but adescription thereof is omitted.

With the configuration described above, the user inputs the maximumexposure time Tmax in advance to the exposure-time input unit 167. Bydoing so, the exposure time can be reduced. Thus, image acquisition canbe made more efficient, and it is possible to efficiently acquire imageswith a wider dynamic range.

This embodiment has been described in terms of an example in which theuser sets the maximum exposure time. However, the time input to theexposure-time input unit 167 may be just the actual exposure time. Inother words, it is not limited to the maximum exposure time.

Third Embodiment

Next, a third embodiment will be described with reference to FIGS. 14and 15.

The basic configuration of the microscope imaging apparatus of thisembodiment is the same as that in the first embodiment, but theconfiguration of the image-acquisition unit is different from the firstembodiment. Therefore, in this embodiment only the image-acquisitionunit will be described using FIGS. 14 and 15, and a description of theillumination unit and so on shall be omitted.

FIG. 14 shows the overall configuration of a microscope imagingapparatus 210 in this embodiment.

As shown in FIG. 14, the microscope imaging apparatus 210 includes astage 30, an illumination unit 40, and an image-acquisition unit 260.The stage 30 holds a specimen 20 and is moveable. The illumination unit40 irradiates the specimen 20 with illumination light. Theimage-acquisition unit 260 acquires signal light emitted from the regionirradiated with the illumination light and measures it.

An imaging lens 61 and a detector 262 are provided in theimage-acquisition unit 260. Return light from the specimen 20 isincident on the imaging lens 61, and the detector 262 detects the returnlight from the specimen 20. An imaging device 263 is disposed in thedetector 262. The imaging device 263 has an anti-smear function and iscapable of acquiring images on the basis of the TDI method. As describedabove, the TDI method is a technique in which, according to the motionof the stage 30, the signal charge in each horizontal line isaccumulated in the imaging device 63.

Smearing is a phenomenon occurring in photoelectric conversion deviceslike CCDs (charge coupled devices). When an excessive signal above acertain level is input to a photoelectric conversion device, thisphenomenon produces a false signal as a result of completely changingthe signal in the longitudinal or transverse direction during signalprocessing.

That is, smearing is a phenomenon whereby, when a highly bright spot oflight, for example, is incident on a photoelectric conversion device, abright band of light extending in the longitudinal or transversedirection is produced.

It is possible to prevent the occurrence of smearing with theconfiguration described above. For example, as shown in FIG. 15, a cellBC of high brightness and a cell DC of low brightness are adjacent toeach other. In this case, if the exposure time is long enough for thelow-brightness cell (biological specimen) DC, smearing occurs in theimage of the high-brightness cell BC. However, with the configurationdescribed above, even if image acquisition is carried out with a longexposure time, which is suitable for the low-brightness cell DC, it ispossible to prevent the occurrence of smearing. Therefore, it ispossible to accurately measure both the high-brightness cell BC and thelow-brightness cell DC.

When using an imaging device not having the anti-smear function, asdescribed above, there is a risk of causing smearing at thelow-brightness cell DC due to the high-brightness cell BC. As a result,it may not be possible to accurately measure the low-brightness cell DC.

With the microscope imaging apparatus of this embodiment, when acquiringa plurality of images of the object under examination, it is possible toacquire images with different exposure times. Then, these images arecombined into a single image. By doing so, it is possible to accuratelyperform measurement even for an examination object having significantbrightness variation.

Fourth Embodiment

Next, a fourth embodiment will be described. The microscope imagingapparatus shown in FIG. 4 is also used in this embodiment. Themeasurement procedure shall be described using FIG. 16.

FIG. 16 is a flowchart showing the measurement procedure in thisembodiment.

First, when measurement commences, prescan preparation is carried out(step S201). In this step, the stage 30 moves to a measurement startingpoint (the point where the imaging device 63 and the specimen 20 havethe positional relationship shown in FIG. 6).

Next, the prescan is carried out (step S202). Fluorescence, for example,is emitted from each of the sample located portions 22, and the prescanis performed to acquire the brightness distribution of thisfluorescence. The exposure time used in the prescan is set to besufficiently short so that the signal charge accumulated in the imagingdevice 63 is not saturated. As described above, the movement speed ofthe stage 30 is set according to the TDI line transfer rate.

FIG. 17 is graph showing the relationship between the prescan brightnessand the exposure time in the main measurement.

When the prescan is completed, preparation for the main measurement ofthe specimen 20 is carried out (step S203). In this step, the stage 30is moved back to the measurement starting point.

The brightness in each sample located portion 22 has already beenacquired in the prescan (step S202). Therefore, from among the acquiredbrightnesses, the maximum value, that is, the maximum intensity value(count), is found. This maximum intensity value is the digital valueoutput from the imaging device 63.

Then, on the basis of the maximum intensity value, the exposure time foreach sample located portion 22 in the current measurement is determined.The exposure time is determined on the basis of the relationship betweenthe prescan brightness and the exposure time for the main measurement,as shown in FIG. 17. The relationship shown in FIG. 17 is obtained inadvance before measurement. (For example, the relationship shown in FIG.17 is obtained by measurement or simulation.)

Once the exposure time has been determined, the main measurement (imageacquisition) of the specimen 20 is carried out (step S204). In thisstep, the image of each sample located portion 22 is acquired on thebasis of the exposure time found in the main-measurement preparationstep (step S203). As described above, the exposure time is expressed bythe TDI line transfer rate and the cumulative number of charged pixels.Also, the stage 30 is moved at a speed in synchronization with the TDIline transfer rate.

In this embodiment too, instead of the imaging device 63 describedabove, an imaging device 63 a having an electronic shutter may be used.Use of such an imaging device 6.3 a is beneficial in that it is possibleto reduce the exposure time. The electronic shutter and the mechanicalshutter are as described previously.

The TDI method is also used in this embodiment. The timing chart for theelectronic shutter used in this TDI method is as shown in FIG. 9.

With the configuration described above, it is possible to obtain asuitable exposure time for measurement of the specimen 20 on the basisof the prescan performed before measurement of the specimen 20.Accordingly, on the basis of the prescan before measurement, a suitableexposure time can be determined even when measuring a specimen 20 whosebrightness changes with time. As a result, the specimen can beaccurately measured.

More concretely, setting an exposure time longer than the appropriatetime can prevent the problem of saturation of the accumulated charge.Also, setting an exposure time shorter than the appropriate time canprevent the problem of insufficient charge accumulation (onlyaccumulating the noise level).

FIG. 18 shows an example of the configuration of another microscopeimaging apparatus 200. As shown in FIG. 18, for example, a light controlmember 70 may be disposed between the imaging lens 61 and the detector62 in the image-acquisition unit 60. For example, a neutral density (ND)filter or an electro-micro mirror device whose transmittance is variedby application of an electric potential may be used as the light controlmember 70.

With the configuration described above, the transmittance of the lightcontrol member 70 can be adjusted according to the exposure timeobtained in the prescan. Thus, the range of possible exposure times formeasurement can be increased. As a result, it is possible to accuratelycarry out measurement even for a specimen 20 having high brightness orlow brightness.

Fifth Embodiment

Next, a fifth embodiment will be described with reference to FIG. 19.

The basic configuration of the microscope imaging apparatus of thisembodiment is the same as that of the fourth embodiment, but the prescanmethod is different from that in the fourth embodiment. Therefore, inthis embodiment, only the prescan method is described using FIG. 19, anda description of the main measurement method and so on is omitted.

FIG. 19 shows the overall configuration of a microscope imagingapparatus 210 of this embodiment.

As shown in FIG. 19, the microscope imaging apparatus 210 includes astage 30, an illumination unit 140, and an image-acquisition unit 60.The stage 30 holds a specimen 20 and is moveable. The illumination unit140 irradiates the specimen 20 with illumination light. Theimage-acquisition unit 60 acquires signal light emitted from the regionirradiated with the illumination light and measures it.

A light source lamp 41, a collector lens 42, a reflecting mirror 43, afield stop 44, a lens 45, an aperture stop 46, and a mirror 47 areprovided in the illumination unit 140. The collector lens 42 collectslight emitted from the lamp 41. The reflecting mirror 43 reflectsbackward-propagating light back to the lamp 41. The diameter of theopening of the field stop 44 can be adjusted. The lens 45 images thefield stop 44 at infinity. The diameter of the opening of the aperturestop 46 can be adjusted. The mirror 47 selectively transmits light. Anobjective lens 149 forms an image of the specimen 20; however, since theobjective lens 149 focuses the light from the lamp 41 on the specimen20, it can also be considered part of the illumination unit 140.

A low-magnification objective lens 149A used in the prescan and ahigh-magnification objective lens 149B used in the main measurement areused as the objective lens 149. These objective lenses 149A and 149B areretained by a known switching mechanism such as a revolver or the like.Thus, it is possible to change the objective lens as required.

With the configuration described above, since it is possible to acquirea wide-field image during the prescan, the prescan time can beshortened.

For example, the high-magnification objective lens 149B used in the mainmeasurement is a 20× lens, and the low-magnification objective lens 149Aused in the prescan is a 4× lens. Thus, because the pixel pitch on thestage 30 is five times larger, the stage speed can be increased by afactor of five. Furthermore, because the field is five times wider, thenumber of line scans can be reduced to one fifth. Therefore, compared tothe case where the prescan and the main measurement are carried out withthe same magnification, the prescan time required for the entire surfaceof the specimen 20 can be reduced to 1/25.

Also, the light (brightness) from the specimen may vary over time,depending on the specimen. In such a case, the appropriate exposure timealso changes over time. Therefore, if the time required for the prescancan be reduced, as in this embodiment, it is possible to reduce the timeinterval from the prescan to the main measurement. As a result, it ispossible to carry out measurement with the appropriate exposure time.

Sixth Embodiment

Next, a sixth embodiment will be described with reference to FIG. 20.

The basic configuration of the microscope imaging apparatus of thisembodiment is the same as that of the fourth embodiment, but theimage-acquisition unit is different from that in the fourth embodiment.Therefore, in this embodiment, only the image-acquisition unit isdescribed using FIG. 20, and a description of the illumination unit andso on is omitted.

FIG. 20 shows the overall configuration of a microscope imagingapparatus 220 of this embodiment.

As shown in FIG. 20, the microscope imaging apparatus 220 includes astage 30, an illumination unit 40, and an image-acquisition unit 260.The stage 30 holds a specimen 20 and is moveable. The illumination unit40 irradiates the specimen 20 with illumination light. Theimage-acquisition unit 260 acquires signal light emitted from the regionirradiates with the illumination light and measures it.

An imaging lens 61, a detector 62, and a scaling lens 261 are providedin the image-acquisition unit 260. Return light from the specimen 20 isincident on the imaging lens 61. The detector 62 detects the returnlight from the specimen 20. The scaling lens 261 can acquire images overa wide field.

The scaling lens 261 is inserted into the light path between theobjective lens 49 and the detector 62. It may be capable of beinginserted in and removed from the light path. Also, the scaling lens 261may be retained by a known switching mechanism such as a revolver. Bydoing so, it is possible to switch between scaling lenses with differentmagnifications, as required.

With the configuration described above, by inserting the scaling lens261 into the light path between the objective lens 49 and the detector62 during the prescan, it is possible to acquire images over a widefield of view. Accordingly, the prescan time can be reduced.

Furthermore, since it is possible to reduce the time interval from theprescan to the main measurement, even for a specimen 20 whose brightnessvaries with time, it is possible to carry out measurement with theappropriate exposure time.

Seventh Embodiment

Next, a seventh embodiment will be described with reference to FIGS. 21and 22.

The basic configuration of the microscope imaging apparatus of thisembodiment is the same as that of the fourth embodiment, but the methodof controlling the exposure time is different from that in the fourthembodiment. Therefore, only the method of controlling the exposure timeis described using FIGS. 21 and 22, and a description of the structureand so on is omitted.

FIG. 21 shows the overall configuration of a microscope imagingapparatus 230 in this embodiment. FIG. 22 is a flowchart showing themeasurement procedure in this embodiment.

As shown in FIG. 21, the microscope imaging apparatus 230 includes astage 30, an illumination unit 40, and an image-acquisition unit 360.The stage 30 holds a specimen 20 and is moveable. The illumination unit40 irradiates the specimen 20 with illumination light. Theimage-acquisition unit 360 acquires signal light emitted from the regionirradiated with the illumination light and measures it.

An imaging lens 61 and a detector 62 are provided in theimage-acquisition unit 360. Return light from the specimen 20 isincident on the imaging lens 61. The detector 62 detects the returnlight from the specimen 20.

The detector 62 is connected to an image processing unit 64, whichprocesses the output from the detector 62. A monitor 65 for displayingthe processed signal is connected to the image processing unit 64. Astage driving mechanism 31 is connected to a computer 366.

The computer 366 includes a maximum-exposure-time input unit 367, acalculating unit 368, and a selecting unit 369. A maximum exposure timeTmax for measuring the specimen 20 is input to the maximum-exposure-timeinput unit 367. The maximum exposure time Tmax is set by the user. Thecalculating unit 368 calculates an exposure time Tpre on the basis ofinformation obtained in a prescan. The selecting unit 369 carries outcomparison and selection of the exposure time Tpre and the maximumexposure time Tmax.

Next, the measurement procedure will be described.

As shown in FIG. 22, first, the user inputs the maximum exposure timeTmax for the main measurment (step S211). Then, measurement commences.

Next, prescan preparation is carried out (step S212). In this step, thestage 30 moves to a measurement starting point (the point where theimaging device 63 and the specimen 20 have the positional relationshipshown in FIG. 6).

Next, the prescan is performed (step S213). Fluorescence, for example,is emitted from each sample located portion 22, and the prescan isperformed to acquire brightness distribution information of thisfluorescence. The exposure time in the prescan is set to a sufficientlyshort duration so that, for example, the signal charge accumulated inthe imaging device 63 is not saturated. Also, as described above, themovement speed of the stage 30 is set according to the TDI line transferrate.

Once the prescan has been completed, preparation for the mainmeasurement of the specimen 20 is carried out (step S214). In this step,the stage 30 is moved back to the measurement starting point.

The brightness in each sample located portion 22 has already beenacquired in the prescan (S213). Thus, a maximum value, that is, amaximum intensity value (count), is found from among the acquiredbrightnesses. This maximum intensity value is a digital value outputfrom the imaging device 63.

Then, the exposure time Tpre for each sample located portion 22 isdetermined on the basis of the maximum intensity value found. Theexposure time Tpre is determined on the basis of the relationshipbetween the prescan brightness and the exposure time in the mainmeasurement, as shown in FIG. 17. The relationship shown in FIG. 17 isdetermined in advance before the measurement. (For example, therelationship shown in FIG. 17 is determined by measurement orsimulation.)

Here, the maximum exposure time Tmax and the exposure time Tpre arecompared. On the basis of the comparison result, if the exposure timeTpre is shorter than the maximum exposure time Tmax, the exposure timeTpre is used as the exposure time for the main measurement. On the otherhand, if the exposure time Tpre is longer than the maximum exposure timeTmax, the maximum exposure time Tmax is used as the exposure time in themain measurement.

When the exposure time has been determined, the main measurement (imageacquisition) of the specimen 20 is carried out (S215). In this step, animage of each sample located portion 22 is acquired on the basis of theexposure time determined in the main measurement preparation (stepS214). As described above, the exposure time is given by the TDI linetransfer rate and the cumulative number of charged pixels. Also, thestage 30 is moved at a speed in synchronization with the TDI linetransfer rate.

By setting the exposure time as described above, it is possible toprevent the exposure time from becoming too long when carrying out themain measurement of a specimen 20 having low brightness. Moreover, it ispossible to measure a specimen 20 whose brightness varies over timeusing the appropriate exposure time.

Eighth Embodiment

Next, an eighth embodiment will be described with reference to FIGS. 23Aand 23B.

The basic configuration of the microscope imaging apparatus of thisembodiment is the same as that in the fourth embodiment, but thespecimen is different from that in the fourth embodiment. Therefore,only the specimen is described using FIGS. 23A and 23B, and adescription of the illumination unit and so on is omitted.

FIGS. 23A and 23B show the configuration of a specimen 20 in thisembodiment.

As shown in FIG. 23A, the specimen 20 is disposed so that the brightnessin the sample located portions 22 arrayed in the X direction (the samplelocated portions 22 enclosed by the ellipses in FIG. 23A) issubstantially uniform.

With the configuration described above, by performing the prescan in onerow in the Y direction in FIG. 23A, it is possible to obtain anappropriate exposure time. Accordingly, the time required for theprescan can be reduced.

The specimen 20 may be arranged as shown in FIG. 23A. Also, as shown inFIG. 23B, sample located portions 22 having substantially uniformbrightness may be arranged so as to be grouped in regions formed of twoor three rows in the X direction and two rows in the Y direction (theregion enclosed by the ellipses in FIG. 23B).

By adopting such an arrangement, as shown in FIG. 23B, it is possible toachieve the appropriate exposure time by performing the prescan in tworows. Therefore, the time required for the prescan can be reduced.

The arrangement of the sample located portions 22 having substantiallyuniform brightness is not particularly limited; any arrangement ispossible so long as the number of prescans can be reduced.

With the microscope imaging apparatus of this embodiment, it is possibleto achieve an appropriate exposure time for measuring the object underexamination on the basis of the prescan performed before the mainmeasurement. Therefore, a specimen whose brightness varies over time canbe accurately measured.

Ninth Embodiment

A ninth embodiment will now be described. FIG. 24 shows the overallconfiguration of a microscope imaging apparatus 310 of this embodiment.Components that are the same as those in FIG. 4 are assigned the samereference numeral, and a description thereof shall be omitted.

With the microscope imaging apparatus 310 of this embodiment, it ispossible to acquire images by the TDI method and by a two-dimensionalimaging method.

An imaging lens 61 and a detector 62 are provided in animage-acquisition unit 60. Return light (fluorescence) from the specimen20 is incident on the imaging lens 61. The imaging lens 61 focuses(images) the incident light at a predetermined position. The detector 62is disposed at the predetermined position and detects the return lightfrom the specimen 20. An imaging device 63 that is capable of the TDImethod and the two-dimensional imaging method is provided in thedetector 62.

The output from the detector 62 is connected to an image processing unit64 that processes the signal output from the detector 62. A monitor 65for displaying the processed signal is connected to the image processingunit 64. The image processing unit 64 is connected to a computer 66. Amirror driving mechanism 48 and a stage driving mechanism 31 are alsoconnected to the computer 66.

The computer 66 is provided with a specimen-parameter input unit(examination-object-parameter input unit) 67, a calculating unit 68, anda switching unit 69. The specimen-parameter input unit 67 obtains andinputs information on the specimen 20 in advance. The calculation unit68 calculates the scanning time for the stage 30 on the basis of thespecimen parameters output from the specimen-parameter input unit 67.The switching unit 69 switches between the TDI method and thetwo-dimensional imaging method on the basis of the calculation resultsfrom the calculation unit 68, and output a switching signal to thedetector 62.

The calculation unit 68 calculates the scanning time for the stage 30 inthe TDI method and the two-dimensional imaging method. Then, bycomparing the durations of the scanning times used in each method, itselects the method having the shorter scanning time.

In this way, the measurement carried out by the microscope imagingapparatus 310 is performed while scanning the plurality of samplelocated portions 22, that is, while scanning the cells in thetwo-dimensional patterned portion 23. Accordingly, when measuring thetwo-dimensional patterned portion 23 with the TDI method or thetwo-dimensional imaging method, the shorter time required for scanning,that is, the shorter measurement time, is employed. The specimen 20 usedhere has the configuration shown in FIGS. 5A and 5B.

The method of estimating the respective scanning times of the specimen20 when the specimen 20 is imaged with the TDI method and thetwo-dimensional imaging method shall be described next.

First, the parameters used in estimating the scanning time of thespecimen 20 will be described.

FIG. 25A shows the parameters for the specimen. FIG. 25B shows theparameters for the imaging device 63.

As shown in FIG. 25A, a measurement substrate 21 has a rectangular outershape. The length in the X direction is A (mm), and the length in the Ydirection is B (mm). Also, the sample located portions 22 are arrangedin a CxD array, where C is the number in the X direction and D is thenumber in the Y direction. Therefore, the total number of sample locatedportions 22 is C×D.

The exposure time for each sample located portion is E (s), and thenumber of measurement wavelengths is F. For example, when observing twodifferent fluorescence dyes (fluorescence wavelengths), the number ofmeasurement wavelengths F is two.

As shown in FIG. 25B, the pixel pitch of the imaging device 63 is G(mm). The number of pixels disposed in the X direction is H (pixels),and the number of pixels disposed in the Y direction is I (pixels).Furthermore, the magnification of the objective lens is J1, and theprojection magnification is J2.

When measuring the specimen 20 with the TDI method, as shown in FIG. 6,the entire surface of the two-dimensional patterned portion 23 isscanned regardless of whether or not the sample located portions 22exist.

When measuring fluorescence with a plurality of wavelengths, the numberof wavelengths that can be observed in one scan is only one. Therefore,the stage must be moved back and forth corresponding to the number ofwavelengths. For example, when measuring fluorescence with twowavelengths, to acquire an image of one line, the stage 30 must be movedback and forth twice.

The calculation for estimating the measurement time in the TDI method isdescribed below.

The product of the TDI line transfer rate and the number of pixels inthe imaging device 63 in the transfer direction gives the exposure time.Therefore, the transfer rate (Tshift) is given by:Tshift=E/I (seconds).

The stage speed (Vstage) can be expressed by the ratio of the pixel sizeon the examination surface to the transfer rate. Therefore, the stagespeed is expressed by:Vstage=(G/(J1×J2))/Tshift (mm/sec).

The time required for a one-way scan of the measurement substrate 21(Tsingle) is given by:Tsingle=B/Vstage (seconds).

Detection is not performed when returning the stage 30, but it isassumed that the same time is required as for the one-way scan of thestage 30. In such a case, the time required for one reciprocation of thestage 30 (Tdouble) is expressed as:Tdouble=2×Tsingle (seconds).

When measuring fluorescence with a plurality of wavelengths, the stageis reciprocated according to the number of wavelengths measured.Therefore, the time required to complete acquisition of one line (Tline)is expressed as:Tline=F×Tdouble (seconds).

The number of scans (N) can be expressed as the ratio of the size of themeasurement substrate 21 in the X direction to the size of the imagingdevice 63 in the X direction, on the surface of the object underexamination. Therefore, the number of scans is given by:N=[A/(H×G/J)]+1 (repetitions)

-   -   where the square brackets indicate Gauss' symbol, that is, the        greatest integer that is not over that value, for example,        [2.34]=2.

Therefore, the scanning time in the TDI method (Ttdi) is:Ttdi=C×Tline (seconds).

Instead of the imaging device 63 described above, an imaging device 63 ahaving an electronic shutter may be used in this embodiment too. Usingsuch an imaging device 63 a is advantageous in that the exposure timecan be reduced. The electronic shutter and the mechanical shutter are asdescribed previously.

The timing chart of the electronic shutter in the TDI method is as shownin FIG. 9.

When determining the scanning time of the specimen 20, the maximum TDItransfer rate (t1) is Tshift.

Next, the method of estimating the measurement time in thetwo-dimensional imaging method will be described.

FIG. 26 depicts scanning in the two-dimensional imaging method. In FIG.26, for the sake of simplifying the illustration, the imaging device 63is depicted as moving.

As shown in FIG. 26, when carrying out measurement of the specimen 20with the two-dimensional imaging method, the stage 30 is moved whileperforming measurement. By moving the stage 30, the measurement pointsequentially moves to the next sample located portion 22 in theexamination region.

FIG. 27 is a graph depicting the operation of the stage 30 in thetwo-dimensional imaging method.

As shown in FIG. 27, the stage 30 is driven while alternating betweenacceleration and deceleration. The motion of the sample located portions22 advances in this way. The stage 30 accelerates during the period T1to T2, moves at constant speed during period T2 to T3, deceleratesduring period T3 to T4, and stops during period T4 to T5.

Thus, measurement of the sample located portion 22 is carried out duringperiod T4 to T5 where the stage 30 stops.

In the two-dimensional imaging method, charge transfer in the imagingdevice 63 does not occur, unlike the TDI method. Therefore, in thetwo-dimensional imaging method, image acquisition can be carried outregardless of the direction of motion of the stage 30.

Next, the method of estimating the measurement time in thetwo-dimensional imaging method will be described.

Here, the time to move between the sample located portion 22 is K(seconds). In addition, when measuring a plurality of wavelengths, thedichroic mirror in the microscope is changed. Therefore, the timerequired to change the dichroic mirror is L (seconds). In themicroscope, the dichroic mirrors are normally held in a turret, which isrotated by a driving mechanism such as a motor or the like. Thus,changing of the dichroic mirror is carried out electrically.

The measurement time Tmeasure for one sample located portion 22 dependson the exposure time E (seconds), the time for changing the dichroicmirror L (seconds), and the number of measurement wavelengths F, and isgiven by:Tmeasure=F×E+(F−1)×L (seconds).

Considering the period for moving between the sample located portions22, the measurement interval Twell between one sample located portion 22and another sample located portion 22 is given by:Twell=Tmeasure+K (seconds).

Also, because the sample located portions 22 are arranged in a C×Dmatrix, the scanning time for the two-dimensional imaging method (T2D)is:T2D=C×D×Twell (seconds).

Next, an example of an actual estimation is shown in Tables 1 to 3.

Table 1 shows values of parameters used in the estimation. The values ofparameters C, D, E, and F in Table 1 are shown in Table 2. Table 2 showsthe estimated results of the measurement time corresponding to theparameters C, D, E, and F. Tables 3 and 4 show intermediate results ofthe estimation for each parameter. TABLE I Switching time forMeasurement No. measure- Magnification two-dimensional substrate sizeNo. wells ment wave- No. pixels Objective Projection imaging method X YX Y Exposure lengths Pixel X Y lens (magnifi- lens (magnifi- MovingSwitching (mm) (mm) (no.) (no.) time (s) Wavelength size (mm) pixelspixels cation) cation) time (s) time (s) A B C D E F G H I J1 J2 K L 2676 parameters 0.0065 1300 1000 20 0.5 4 1

TABLE 2 Measurement time(excluding Measurement conditions image transfertime) No. sample Exposure No. TDI Two-dimensional located portions timewavelengths method imaging method C*D (no.) E (s) F Ttdi (s) T2D (s)Method used 1000 0.1 1 725 4100 TDI method 100 0.1 1 725 410Two-dimensional imaging method 1000 0.1 2 1450 5200 TDI method 100 0.1 21450 520 Two-dimensional imaging method

TABLE 3 1 2 3 4 Tshift (s) 1.00E−04 1.00E−04 1.00E−04 1.00E−04 Vstage(s) 6.5 6.5 6.5 6.5 Tstage (s) 11.7 11.7 11.7 11.7 Tdouble (s) 23.4 23.423.4 23.4 Tline (s) 23.4 23.4 46.8 46.8 N (repetitions) 31 31 31 31 Ttdi(s) 725 725 1450 1450

TABLE 4 5 6 7 8 Tmeasure (s) 0.1 0.1 1.2 1.2 Twell (s) 4.1 4.1 5.2 5.2T2D (s) 4100 410 5200 520

FIG. 28 shows an arrangement in which the density of sample locatedportions 22 on the measurement substrate 21 changes depending onposition (that is, the density is non-uniform). FIG. 29 depicts anexample where the scanning method switches between the TDI method andthe two-dimensional imaging method.

As shown in FIG. 28, when the density of the sample located portions 22is non-uniform, two imaging methods are used. Specifically, as shown inFIG. 29, the region where the density of specimen location portions 22is high (the region at the left in FIGS. 28 and 29) is measured with theTDI method, and the region where the density is low (the region at theright in FIGS. 28 and 29) is measured with the two-dimensional imagingmethod. In this way, by switching between the TDI method and thetwo-dimensional imaging method depending on the distribution of samplelocated portions 22, it is possible to select the method having thehigher scanning speed for carrying out measurement. As a result, themeasurement time can be shortened.

In the measurement described above, measurement of the specimen 20 iscarried out right after the measurement preparation has been completed.However, a prescan may be carried out to measure the density of samplelocated portions 22 in the specimen 20 before measuring the specimen 20.Doing so allows the main measurement to be carried out after selectingthe TDI method or the two-dimensional imaging method for measuring thespecimen 20 on the basis of information about the density obtained inthe prescan.

When performing the prescan, it is preferable that the objective lens 49have low magnification. By doing so, the field of view of the microscopeis widened, and therefore, it is possible to complete the prescan in ashorter period of time. Also, user input is not required beforehand.

Furthermore, when the density of sample located portions 22 isnon-uniform, as shown in FIG. 28, it requires a substantial effort forthe user to input information for changing the imaging method.Conversely, since the imaging method can be changed automatically on thebasis of the prescan, no such effort is required.

With the configuration described above, the method having the shorterscanning time among the TDI method and the two-dimensional imagingmethod is selected as the method used in the measurement. Therefore,compared to the case where the method is not selected, the scanning inthe measurement can be carried out more quickly. Also, being able toselect the method having the higher scanning speed allows the timerequired for measuring the specimen 20 to be reduced.

Moreover, it is possible to automatically select either the TDI methodor the two-dimensional imaging method, whichever is most appropriate, onthe basis of the number of measurement wavelengths, the exposure time,and the density of the specimen 20. Therefore, it is easy to speed upthe scanning procedure, which allows the measurement time to be easilyreduced.

With the microscope imaging apparatus of this embodiment, the method formeasuring the object under examination can be selected from either theTDI method or the two-dimensional imaging method on the basis of thescanning time described above, and it is therefore possible to selectthe method having the shorter scanning time.

Accordingly, compared to a case where the imaging method is notselected, the scanning during measurement of the object underexamination can be made faster, and the time required for measurementcan thus be reduced.

Tenth Embodiment

Next, a tenth embodiment will be described with reference to FIGS. 30 to45. The tenth embodiment is a biological-specimen examination systemusing one of the microscope imaging apparatuses according to the firstto ninth embodiments described above.

FIG. 30 is a perspective view showing an overview of abiological-specimen examination system 410 according to this embodiment.FIG. 31 is a schematic diagram showing the system configuration of thebiological-specimen examination system.

As shown in FIGS. 30 and 31, the biological-specimen examination system410 includes a detection unit 420 and a culture unit 470. The detectionunit 420 and the culture unit 470 are preferably disposed adjacent toeach other, and more preferably, the units 420 and 470 are disposed incontact with each other.

As shown in FIGS. 30 and 31, the detection unit 420 includes aninsulated compartment 421 that contains biological specimens and adetection section (microscope imaging apparatus) 440 that measures cellsCE serving as the biological specimens.

The insulated compartment 421 includes a heater 421H for keeping theinterior of the insulated compartment 421 at a predeterminedtemperature; a stage 422 that holds an incubator box 500 (describedlater); a transmission light source (illuminating unit) 423 thatirradiates the cells CE with light; a fan 424 to make the temperatureinside the insulated compartment 421 uniform; a UV lamp 425 forsterilizing the interior of the insulated compartment 421; a carrier 426that covers a culture-fluid circulating tube 477 (described later), aculture-gas supply tube 497, and so on; a door 427 used when putting theincubator box 500 or the like in the insulated compartment 421 or whenremoving it therefrom; and a main power-supply switch 428 for turning onand off the main power supply of the detection unit 420.

The stage 422 includes an X-axis motion stage 422× and a Y-axis motionstage 422Y that move relative to each other in mutually orthogonaldirections, and scanning of the stage 422 is controlled by a stagescanning unit (motion unit) 429.

The stage scanning unit 429 is formed of an X-axis coordinate detectionunit 430 that detects the X-axis coordinate value of the X-axis motionstage 422X, an X-axis scanning controller 431 that controls the motion(scanning) of the X-axis motion stage 422X, a Y-axis coordinatedetection unit 432 that detects the Y-axis coordinate value of theY-axis motion stage 422Y, and a Y-axis scanning controller 433 thatcontrols the motion (scanning) of the Y-axis motion stage 422Y.

The X-axis coordinate detection unit 430 and the Y-axis coordinatedetection unit 432 are configured so as to output the detectedX-coordinate of the X-axis motion stage 422X and the Y-coordinate of theY-axis motion stage 422Y, respectively, to a computer PC. The X-axisscanning controller 431 and the Y-axis scanning controller 433 areconfigured so as to control the scanning of the X-axis motion stage 422Xand the scanning of the Y-axis motion stage 422Y on the basis ofrespective instructions from the computer PC.

The mechanism for driving the X-axis motion stage 422X and the Y-axismotion stage 422Y may be, for example, a combination of a motor and aball screw.

As described above, the computer PC controls the scanning of the X-axismotion stage 422X and the Y-axis motion stage 422Y, and in addition, asdescribed later, it also controls the detection system of the cells CE,performs analysis of the images of the cells CE, and so on and controlsthe X-axis motion stage 422X, the Y-axis motion stage 422Y, thedetection system, and the analysis system in a coordinated fashion.

A condenser lens 434 that focuses the light emitted from thetransmission light source 423 onto the cells CE is disposed between thetransmission light source 423 and the incubator box 500.

A shutter 435 may be disposed between the condenser lens 434 and theincubator box 500, or the shutter 435 may be omitted.

The fan 424 is disposed on the wall of the insulated compartment 421.Operating this fan 424 causes air convection inside the insulatedcompartment 421, which enables the temperature inside the insulatedcompartment 421 to be easily kept uniform and constant.

The UV lamp 425 is connected to a UV-lamp switch 436 disposed on thewall of the detection unit 420, and a control timer 437 thatperiodically operates the UV lamp 425 is disposed between the UV lamp425 and the UV-lamp switch 436. Furthermore, a sterilization-in-progressindicator lamp (not shown) for indicating that the UV lamp 425 is turnedon is provided.

For example, if the UV-lamp switch 436 is pressed when not measuring thecells CE, the timer 437 commences counting and power is supplied to theUV lamp 425 to irradiate the interior of the insulated compartment 421with UV light (ultraviolet light). At the same time, thesterilization-in-progress indicator lamp is illuminated. Then, after apredetermined amount of time (for example, 30 minutes), the timer 437finishes counting, the timer 437 stops supplying power to the UV lamp425, and the UV irradiation is stopped. Also, thesterilization-in-progress lamp is turned off.

The UV lamp 425 can be controlled independently of the main power-supplyswitch 428 so that it can be operated even if the main power-supply isoff.

The illumination time of the UV lamp 425 may be 30 minutes, as describedabove. Alternatively, an illumination time less than 30 minutes or morethan 30 minutes may also be used so long as contamination and so forthinside the insulated compartment 421 can be completely killed.

The door 427 is formed of a metal such as aluminum or the like that hasbeen subjected to anodizing, or it may be formed of a translucent resinwith high opacity.

The door 427 may have a double-layer construction with an air gap, orthe inside may be formed of metal and the outside formed of resin. Usingresin on the outside of the door 427 can prevent heat from inside theinsulated compartment 421 from escaping through the door 427. Also,forming the inside of the door 427 of anodized metal can preventdeterioration of the lifetime of the door 427 due to the UV lamp 425.

If the door 427 has a double-layer construction of metal or metal andresin, since light is completely blocked, it is preferable to provide aninspection window at a position where the incubator box 500 can beviewed. The inspection window is preferably formed of transparent resinor glass, and an openable/closable cover is preferably disposed at theouter side thereof.

As shown in FIGS. 30 and 31, a detection section 440 includes a heater440H for keeping the interior of the detection section 440 at apredetermined temperature; incident light sources 441A and 441B thatirradiate the cells CE from the detection section 440 side; a light-pathswitching unit 442 that switches the light path from the incident lightsources 441A and 441B; a light-intensity adjusting mechanism 443 thatadjusts the intensity of the irradiated light; a lens system 444 thatfocuses the irradiated light towards the cells CE; a filter unit 445that controls the wavelength of the irradiated light and the wavelengthof the detection light; an autofocus (AF) unit 446 that performs afocusing operation with respect to the cells CE; a revolver 447 providedwith a plurality of objective lenses 448 having different magnificationsand properties; a detector (imaging unit) 449 that detects detectionlight from the cells CE; a light-intensity monitor 450 that measures theintensity of the detection light; a fan 451 that makes the temperatureinside the detection section 440 uniform; and a cooling fan 452 thatcools the interior of the detection section 440.

The incident light sources (illumination unit) 441A and 441B, which areformed of mercury lamps, for example, are disposed outside the detectionsection 440 and are connected to a power supply 453 that supplies powerthereto.

Normally, a single incident light source, for example, the incidentlight source 441A, is used; however, if the intensity of the incidentlight source 441A falls below a certain prescribed value, light isirradiated from the other incident light source 441B and the powersupply to the first incident light source 441A is turned off.

The light-path switching unit 442 is configured to guide illuminationlight from either the incident light source 441A or the incident lightsource 441B to the light-intensity adjusting mechanism 443. Also, thelight-path switching unit 442 is provided with a light-path control unit454, which is connected to the computer PC (described later) forcontrolling the light-path switching unit 442 on the basis of aninstruction from the computer PC.

At the emitting side of the light-path switching unit 442 where theillumination light is emitted, a shutter 442S that controls thetransmission and blocking of the illumination light is provided.

The light-intensity adjusting mechanism 443, which is disposed at theemission side of the shutter 442S where the illumination light isemitted, adjusts the intensity of the illumination light passing throughthe shutter 442S. A known aperture mechanism, for example, may be used,or any other known mechanism or technique that can adjust the lightintensity may be used.

The light-intensity adjusting mechanism 443 is provided with alight-intensity control unit 455, which is connected to the computer PC(described later) for controlling the light-intensity adjustingmechanism 443 on the basis of an instruction from the computer PC.

The lens system 444 is disposed at the emission side of thelight-intensity adjusting mechanism 443 where the illumination light isemitted. The lens system 444 includes a pair of lenses 444A and 444B anda stop 444C disposed between the lens 444A and the lens 444B.

The filter unit 445 includes an excitation filter 456, a dichroic mirror457, and an absorption filter 458. The excitation filter 456 is a filterthat transmits wavelengths which contribute to the generation offluorescence in the cells CE (excitation light) from among theillumination light and is disposed so that the illumination lightemitted from the lens system 444 is incident on the excitation filter456. The dichroic mirror 457 is an optical element that splitsexcitation light and fluorescence. More specifically, the dichroicmirror 457 is disposed so as to reflect excitation light transmittedthrough the excitation filter 456 towards the cells CE and to transmitfluorescence from the cells CE. The absorption filter 458 is an opticalelement that separates fluorescence from the cells CE from otherunwanted scattered light. The absorption filter 458 is disposed so thatlight transmitted through the dichroic mirror 457 is incident thereon.

The filter unit 445 is provided with a filter control unit 446C thatcontrols the wavelengths of the excitation light emitted from the filterunit 445 and the detection light (fluorescence) on the basis ofinstructions from the computer PC (described later).

One excitation filter 456, one dichroic mirror 457, and one absorptionfilter 458 may be used, or alternatively, a plurality of each may beused.

The AF unit 446 is disposed at the emission side of the filter unit 445where the excitation light is emitted and is disposed so that theexcitation light is focused onto the cells CE via one of the objectivelenses 448, on the basis of an instruction from the computer PC(described later).

The revolver 447 is disposed at the emission side of the AF unit 446where the excitation light is emitted and is provided with the pluralityof objective lenses 448 having different magnifications. The revolver447 is provided with an objective-lens control unit 459 which selectsand controls the objective lens 448 on which the excitation light isincident, on the basis of an instruction from the computer PC (describedlater).

The objective lenses 448 are configured to allow examination, from thedetection section 440, of the interior of the incubator box inside theinsulated compartment 421 via holes provided in the X-axis motion stage422× and the Y-axis motion stage 422Y.

The holes in the X-axis motion stage 422× and the Y-axis motion stage422Y are large enough to allow viewing over the operating region of thestage, with some additional margin.

Therefore, although the ambient air inside the insulated compartment 421should be kept at a humidity suitable for culturing cells, the ambientair may escape to the detection section 440 through the holes, whichmakes it impossible to maintain the temperature suitable for culturingcells, and therefore, there is a risk of bringing about a reduction incell activity.

Thus, a containment mechanism 449 for suppressing the passage of suchambient air, which is at a temperature suitable for cell culturing,between the insulated compartment 421 and the detection section 440.

The containment mechanism 449 should be capable of suppressing the flowof air while not interfering with the operation of the revolver 447 andthe objective lenses 448. For example, it may be a sheet-like mechanismin which, for example, a sheet formed of a flexible material, such as afilm or transparent sheet, is attached to the perimeter of a holeprovided at the boundary between the insulated compartment 421 and thedetection section 440 and in such a manner that it is draped around theperimeter of the revolver.

A focusing lens 460 that focuses the detection light onto the detector449 and the light-intensity monitor 450 is provided at the emission sideof the filter unit 445 where the detection light is emitted.

A half-mirror 461 that reflects some of the detection light towards thedetector 449 and that transmits the remaining detection light towardsthe light-intensity monitor 450 is provided at the emission side of thefocusing lens 460 where the detection light is emitted.

The detector 449 is disposed at a position where the detection lightreflected from the half mirror 461 is incident thereon. Also, a detectorcalculation unit 462 that calculates a detection signal from thedetector 449 and outputs it to the computer PC (described later) isconnected to the detector 449.

The detector 449 is not particularly limited and may use a line sensor,an area sensor, or both a line sensor and an area sensor.

The light-intensity monitor 450 measures the detection light transmittedthrough the half-mirror 461 and is configured so as to output themeasured value to the computer PC.

The intensity of the detection light may be measured using thelight-intensity monitor 450, as described above, or alternatively, theintensity of the detection light may be measured using an illuminancemeter or a power meter.

The heater 440H controls the temperature inside the detection section440 to be from 30° C. to 37° C. The fan 451 is disposed to cause airconvection inside the detection section 440 to make the temperatureinside the detection section 440 uniform. Therefore, the temperatureinside the detection section 440 can be maintained close to thetemperature in the insulated compartment 421, and the temperature in theinsulated compartment 421 can thus be more easily stabilized.

The cooling fan 452 is operated to reduce the temperature inside thedetection section 440 on the basis of the output from a temperaturesensor (not shown) provided inside the detection section 440. Therefore,it is possible to prevent an abnormal rise in temperature inside thedetection section 440 due to heating by, for example, the motors and soforth.

FIG. 32 is a perspective view of the incubator box according to thisembodiment, and FIG. 33 is a cross-sectional view of a chamber accordingto this embodiment.

As shown in FIGS. 32 and 33, the incubator box 500 includes a frame 501,containing a chamber (object under examination) 510, and a cover 502that forms a sealed space together with the frame 501. The frame 501 andthe cover 502 are subjected to magnetic-shielding treatment for blockingexternal magnetic fields and anti-static treatment for eliminating thebuild-up of static electricity in the incubator box 500.

The frame 501 is formed of a base plate 503 and side walls 504, and aregion corresponding to the measurement area of the base plate 503 isformed of a transparent material, such as glass. The other regions ofthe base plate 503 and the side walls 504 are preferably formed of ananti-corrosive material having high opacity, such as anodized aluminumor stainless steel, like SUS316. More preferably, from the viewpoint ofmaintaining the temperature, a material having a low thermalconductivity may be selected.

An adaptor 505 for holding the chamber 510 and a temperature sensor 506for measuring the temperature of the chamber 510 are provided on thebase plate 503. The chamber 510 may be held using the adaptor 505, asdescribed above, or the chamber 510 may be held without using theadaptor 505.

The output from the temperature sensor 506 is input to the computer PCvia an incubator-temperature detection unit 506S and is also input to atemperature-display unit 507 disposed on the wall of the detection unit420. The computer PC controls the heater 421H and so on via anincubator-temperature control unit 506C shown in FIG. 31 to control thetemperature inside the incubator box 500 in order to keep it constant.

The cover 502 includes a glass plate 517 that transmits the illuminationlight and a support portion 517A that supports the glass plate 517. Ananti-reflection film may be formed on both sides of the glass plate 517in a region corresponding to the measurement area. Forming such ananti-reflection film on both sides allows prevention of reflection bythe glass plate 517 during transmission examination and incidenceexamination.

The area of the glass plate 517 may be substantially the same as that ofthe base plate 503 of the incubator box 500, or it may be the minimumnecessary area that does not cause any problem during measurement.

As shown in FIG. 33, the chamber 510 is formed of a lower glass member511 for observation with the objective lens 448, an upper glass member512 for transmitting light from the transmission light source 423, and aframe member 513 that supports the lower glass member 511 and the upperglass member 512.

Joints 514 having channels formed therein for circulating culture fluidare formed at opposing sides of the frame member 513. A culture-fluidcirculating tube 477 (described later) is connected to the joints 514,for allowing culture fluid to circulate between the culture unit 470 andthe detection unit 420.

A pair of flow smoothers 515 for making the flow of culture fluiduniform are disposed in the frame member 513 so as to be substantiallyorthogonal to the flow of culture fluid. The flow smoothers 515 areformed of sheet members in which, for example, small holes are formed ina matrix, and by splitting the culture fluid and flowing it through theplurality of small holes, the flow becomes uniform. A slide glass 516 onwhich the cells CE are disposed is provided between the two flowsmoothers 515.

As described above, the incubator box 500 may be provided with thechamber 510 inside, or, as shown in FIG. 34A, a microplate (object underexamination) 520 (or a well plate) may be disposed inside.

In this configuration, as shown in FIG. 34A, the frame 501 of anincubator box 500 a is provided with a square-shaped reservoir 521surrounding the microplate 520, an internal fan 522 disposed at theinner side of the reservoir 521, a connector 523 for supplying culturegas, and a culture-gas concentration sensor 524 for detecting theconcentration of carbon dioxide in the culture gas.

The temperature sensor 506 is disposed so as to measure the temperatureof the microplate 520. The microplate temperature input to the computerPC from the temperature sensor 506 is collected in the form of text datain a memory and can be subjected to data processing in the computer PC.

The culture-gas concentration sensor 524 outputs the carbon-dioxideconcentration to the computer PC and to a culture-gas concentrationdisplay unit 524D.

The height of side walls 521W of the reservoir 521 is formed to be lowerthan the height of the side walls of the frame 501. Also, the positionalrelationship with respect to the connector 523 is adjusted so that thesupplied culture gas blows against the side walls 521W. Sterilized wateris stored in the reservoir 521, and the humidity inside the incubatorbox 500 a is regulated at about 100%.

The internal fan 522 is disposed so that the microplate 520 is notpositioned in the blowing direction thereof and so that it blows alongthe side walls 521W of the reservoir 521.

The culture-gas concentration sensor 524 may be disposed on the innersurface of one of the side walls 521W of the reservoir 521.Alternatively, the tubes from the incubator box 500 a may be disposedoutside and the culture gas inside the incubator box 500 a may beevacuated with a suction pump to detect the concentration thereof withthe culture-gas concentration sensor 524.

When using this kind of incubator box 500 a, the destination of theculture gas supplied from a culture-gas mixing tank 491 (describedlater) is changed from a culture fluid vessel 472 to the incubator box500 a, and because it is not necessary to supply culture fluid from theculture unit 470, the operation of a culture-fluid pump 480 or the likecan be stopped.

With such a construction, since the humidity environment and culture-gasconcentration in the incubator box 500 a are maintained so that littledamage is caused to the cells CE by changes and non-uniformity in thehumidity and culture-gas concentration, damage to the cells CE can bereduced compared to a thermal environment.

Also, because the humidity and culture-gas concentration in theincubator box 500 a, which is not directly in contact with the detectionsection 440, are maintained, it is possible to prevent contaminationduring examination.

Furthermore, since it is not necessary to maintain the proper humidityand culture-gas concentration when culturing the cells CE in theinsulated compartment 421, the performance of the objective lenses 448and so on disposed in the insulated compartment 421 can be preventedfrom deteriorating. Thus, a reduction in lifetime of the objectivelenses 448 and so on can be prevented.

The chamber 510 may be a sealed enclosure, as described above, or it maybe an open chamber that is not sealed. Such an open chamber is formedwith the same construction as the chamber 510 except for the provisionof the upper glass member 512.

When using such an open chamber, using the incubator box 500 describedabove, the incubator box 500 a is filled with the culture gas and theopen chamber is supplied with culture liquid.

As shown in FIG. 34B, the chamber 510 described above may also be usedin the incubator box 500 a. In such a case, the connector 523 forsupplying culture gas is blocked off, and the culture-gas concentrationsensor 524 is not used. If the size of the chamber 510 is different fromthat of the microplate 520, the chamber 510 may be placed in theincubator box 500 a using an adaptor 505. Sterilized water need not beplaced in the reservoir 521; in fact, the reservoir 521 itself may beeliminated from the incubator box 500 a. Also, the temperature sensor506 measures the temperature in the chamber 510.

The connector 523 may be blocked off, as described above, or it may beleft connected to the culture-gas supply tube 497 and the supply ofculture gas to the incubator box 500 a simply stopped.

As shown in FIGS. 30 and 31, the culture unit 470 includes a sterilizedcompartment 471 containing the culture fluid and a mixing section 490for producing the culture gas.

The sterile compartment 471 includes a heater 471H for keeping theinterior of the sterile compartment 471 at a predetermined temperature;a culture-fluid vessel 472 for storing the culture fluid; an auxiliarytank 473 for storing spare culture fluid; a waste tank 474 into whichused culture fluid is discharged; a UV lamp 425 for sterilizing theinterior of the sterile compartment 471; a door 475 used when puttingthe culture-liquid vessel 472 into the sterile compartment 471 and whenremoving it therefrom; and a main power-supply switch 476 for turning onand off the main power supply for the culture unit 470.

The culture-fluid vessel 472 is provided with a culture-fluidcirculating tube 477 for circulating culture fluid between theculture-fluid vessel 472 and the incubator box 500; a supply tube 478for supplying spare culture fluid from the auxiliary tank 473; and awaste tube 479 for discharging used culture fluid from the culture-fluidvessel 472 to the waste tank 474.

A culture-fluid pump 480 for delivering culture fluid from theculture-fluid vessel 472 to the incubator box 500 and circulating theculture fluid is provided for the culture-fluid circulating tube 477.Using the culture-fluid pump 480, it is possible to replace the culturefluid in the chamber 510 with fresh culture fluid, and therefore, thecells CE can be cultured for a longer period of time compared to a casewhere the culture fluid is not replaced.

A supply pump 481 for transferring culture fluid from the auxiliary tank473 to the culture-fluid vessel 472 is provided for the supply tube 478.In addition, a waste pump 482 for transferring the used culture fluidfrom the culture-fluid vessel 472 to the waste tank 474 is provided forthe waste tube 479.

As described above, the waste tank 474 for storing the used culturefluid may be used. Alternatively, instead of using the waste tank 474, adischarge port for directly discharging the used culture fluid may beprovided.

A culture-fluid temperature sensor (not shown) for detecting theculture-fluid temperature is provided in the culture-fluid vessel 472,and the output from the culture-fluid temperature sensor is input to thecomputer PC via a culture-fluid temperature detector 483. Dataconcerning the culture-fluid temperature input to the computer PC iscollected in a memory in the form of text data and is used whencomparing and verifying the detection results of the cells CE.

The heater 471H is provided with a culture-fluid temperature controller484 that controls the temperature of the culture fluid via thetemperature inside the sterilized compartment 471, on the basis of aninstruction from the computer PC. The temperature of the culture fluidsupplied from the culture-fluid vessel 472 is held at about 37° C. bythe culture-fluid temperature controller 484, which prevents theactivity of the cells CE from dropping due to temperature changes of theculture fluid. Also, a temperature display unit 485 for displaying theculture-fluid temperature detected by the culture-fluid temperaturesensor is provided on the wall of the culture unit 470.

The culture-fluid pump 480 is provided with a culture-fluid-pumpcontroller 486 for controlling the circulation of the culture fluid onthe basis of an instruction from the computer PC. The operation of thesupply pump 481 and the waste pump 482 is also controlled on the basisof instructions from the computer PC.

The UV lamp 425 is connected to a UV-lamp switch 436 disposed on thewall of the culture unit 470, and a timer 437 for periodicallycontrolling the operation of the UV lamp 425 is provided between the UVlamp 425 and the UV-lamp switch 436. Furthermore, asterilization-in-progress indicator lamp (not shown) for indicating thatthe UV lamp 425 is illuminated is also provided.

The UV lamp 425 is controlled independently of the main power-supplyswitch 476 and can be operated even when the main power-supply switch476 is off.

As shown in FIGS. 30 and 31, the mixing section 490 includes a heater(not shown) for keeping the interior of the mixing section 490 at apredetermined temperature; a culture-gas mixing tank 491 for adjustingthe carbon-dioxide concentration in the culture gas supplied to theincubator box 500; and a CO₂-pump 493 for supplying carbon dioxide froma CO₂ tank 492 provided outside the culture unit 470 to the culture-gasmixing tank 491.

A CO₂-concentration detector 494 is provided in the culture-gas mixingtank 491 for detecting the concentration of carbon dioxide therein, andthe output from the CO₂-concentration detector 494 is input to thecomputer PC. The CO₂ pump 493 is provided with a CO₂-concentrationcontroller 495 for controlling the amount of carbon dioxide supplied tothe culture-gas mixing tank 491 on the basis of an instruction from thecomputer PC. Also, a CO₂-concentration display unit 496 for displayingthe carbon-dioxide concentration inside the culture-gas mixing tank 491,which is detected by the CO₂-concentration detector 494, is provided onthe wall of the culture unit 470.

Furthermore, a culture-gas supply tube 497 is provided between theculture-gas mixing tank 491 and the culture-fluid vessel 472.Accordingly, culture gas is supplied to the culture fluid via theculture-gas supply tube 497, which allows a sufficient level of culturegas to be dissolved in the culture fluid. In this way, by producingculture fluid in which culture gas having a 5% concentration of carbondioxide is dissolved inside the culture-fluid vessel 472, culture fluidincluding culture gas and nutrients necessary for nourishing the cellsCE is supplied to the chamber 510. Also, by dissolving the culture gasin the culture fluid, the pH and so forth of the culture fluid can beregulated.

The carbon-dioxide concentration input to the computer PC from theCO₂-concentration detector 494 is collected in the memory in the form oftext data, and data processing can be carried out in the computer PC.

Next, an examination method used in the biological-specimen examinationsystem 410 having the above-described configuration will be described.

First, the scanning method and selection of a detection region in thisembodiment will be described with reference to FIGS. 35A to 35D.

FIGS. 35A to 35D depict examples of the scanning method and selection ofthe detection areas in this embodiment.

In the example shown in FIG. 35A, a measurement region M (the regionsurrounded by the dashed line in the figure) is set by specifying anupper-left point a and a lower-right point b defining the measurementregion M in the displayed image. More concretely, the measurement regionM may be set by dragging a device like a mouse from point a to point b,or it may be specified by inputting the coordinates of the points a andb.

As indicated by the arrows in the figure, regarding the part to bemeasured by the detector 449, the specified measurement region M isscanned from left to right. That is, when scanning from the left to theright in the figure, scanning is performed parallel to the X direction,and when scanning from the right to the left, scanning is performeddownward and to the left at an angle. While scanning from left to right,image acquisition of the cells CE is carried out.

FIG. 35B is an example in which two measurement regions M are specifiedby the method described above. First, the two measurement regions MA andMB are specified using the method described above. The measurementregion MA and the measurement region MB are arranged with a certain gaptherebetween in the X direction in the figure, and they are disposed soas to completely overlap in the Y direction.

As indicated by the arrows, the part to be measured by the detector 449in this example is scanned so as to measure the measurement regions MAand MB in parallel. That is, when scanning from the left to the right inthe figure, scanning is performed from the measurement region MA to themeasurement region MB, and when scanning from the right to the left,scanning is performed from the measurement region MB to the measurementregion MA.

FIG. 35C is an example in which two measurement regions are set usingthe method described above. The two measurement regions MA and MB aredisposed at different positions. Here, the measurement region MA and themeasurement region MB are arranged with a gap therebetween in the Xdirection in the figure, and they are disposed so that they partiallyoverlap in the Y direction in the figure.

As indicated by the arrows in the figure, regarding the parts to bemeasured by the detector 449 in this example, only the portions of themeasurement regions MA and MB that overlap in the Y direction aresequentially scanned. That is, first the non-overlapping portion of themeasurement region MA is scanned. Next, the overlapping portions of themeasurement regions MA and MB are sequentially scanned. Then, thenon-overlapping portion of the measurement region MB is scanned.

FIG. 35D is an example in which two measurement regions M are set by themethod described above. In this example, the two measurement regions MAand MB are the same as those in FIG. 35B, but the scanning method isdifferent.

As indicated by the arrows in the figure, regarding the parts to bemeasured by the detector 449 in this example, the measurement regions MAand MB are scanned independently. That is, after first scanning theentire measurement region MA, the entire measurement region MB isscanned.

Among the scanning methods shown in FIGS. 35A to 35D described above,the method with the shortest total distance moved or shortest scanningtime is automatically selected by the computer PC on the basis ofspecified parameters and a measurement mode, which are described later.

When acquiring images of the region where the cells are cultured, it ispossible to carry out image acquisition only in the required parts, asnecessary, if that region is formed of a plurality of divided regions(detection regions) by, for example, specifying measurement regions Mthat can be imaged.

For example, if the settings of the computer PC are changed toalternately scan the entire region and predetermined parts of the objectto be scanned, it is possible to observe phenomena unique to biologicalspecimens which occur only for a short time. As one example, in a casewhere scanning of the entire region of an object is normally performedevery 30 minutes, so long as scanning is performed in a predeterminedmeasurement region M where cells of interest exist, it is possible todetermine the occurrence of a specific phenomenon that is exhibited onlyevery 15 minutes in those cells of interest.

In order to scan the desired measurement region M in the required time,the scanning time can be reduced, and the time required to irradiateother cells with light can be reduced.

Next, the procedure for measuring the cells CE will be described using aflowchart.

First, before measuring the cells CE, measurement parameters are set.Therefore, the procedure for setting the measurement parameters will bedescribed with reference to FIG. 36.

FIG. 36 is a flowchart showing the procedure for setting the measurementparameters.

First, the measurement parameters are set (step S21).

Then, default conditions are set (step S22). Here, the conditions setare the culturing conditions and the measurement conditions, forexample, a CO₂ concentration of 5%, a temperature of 37° C., and soforth. These conditions can be changed to predetermined conditions bythe user.

Next, the measurement object is selected (step S23). Measurement objectmeans the container of the cells CE, for example, the microplate 520 orthe slide glass 516.

Next, the measurement mode is selected (step S24). Possible measurementmodes include an area acquisition mode, a line acquisition mode, anautomatic mode, and so on. In the automatic mode, the measurement modehaving the shortest measurement time is automatically selected fromamong the other modes.

Next, the measurement magnification is selected (step S25), and afterthat, the detection wavelength is selected (step S26). The measurementmagnification and the detection wavelength can each be automaticallyselected from among two or more options.

Here, as the method of selecting the detection wavelength, a list offluorescent proteins used, for example, GFP, HC-Red, and so on, isstored in advance in the computer PC, and one of them is selected fromthe stored list. The computer PC automatically selects the mostappropriate excitation filter 456, absorption filter 458, and so on forexamination, on the basis of the selected fluorescent protein. In thisway, specific fluorescence from the cells CE can be detected.

The excitation filter 456, the absorption filter 458, the objective lens448 and so on used for measurement are automatically changed insynchronization with the driving of the X-axis motion stage 422X and theY-axis motion stage 422Y.

Next, the measurement interval is set (step S27).

Then, a preview image is acquired (step S28), and the preview image isdisplayed on the monitor (step S29). In the latter step, the previewimage is displayed on the monitor when the user issues an instructionusing a preview button or the like for instructing display of thepreview image on the monitor. Thus, the user can confirm the previewimage displayed on the monitor.

Next, the measurement region is selected (step S30). After selecting themeasurement region, the preview image may be displayed on the monitoragain to confirm whether the measurement region has been correctlyselected.

Next, a predetermined measurement interval is selected from a pluralityof specified measurement intervals (step S31).

Then, upon pressing a start-measurement switch (not shown; step S32),measurement of the cells CE commences (step S33). If thestart-measurement switch is not pressed, the process stands by until thestart-measurement switch is pressed (step S32).

If the start-measurement switch is not pressed in step S32, the processmay jump back to any predetermined step to allow the measurementparameters to be set again.

After the measurement parameters have been set, measurement of the cellsCE is carried out. Therefore, the procedure for measuring the cells CEwill be described with reference to FIGS. 37 and 38.

FIGS. 37 and 38 are flowcharts showing the measurement procedure.

First, when measurement starts, the measurement region is retrieved(step S41). Then, the magnification is retrieved (step S42), and thedetection wavelength is retrieved (step S43).

Next, the measurement mode is retrieved (step S44). Here, theappropriate stage scanning method is determined on the basis of theretrieved measurement region, magnification, detection wavelength(fluorescence wavelength), and so on. If the measurement mode is set tothe automatic mode, the image acquisition mode is also determined atthis point.

Next, the method of operating the X-axis motion stage 422X and theY-axis motion stage 422Y in accordance with the determined stagescanning mode is analyzed (step S45), and data for the analyzedoperating method (operating data) is saved in a table in the computer PC(step S46).

Thereafter, measurement is carried out using a different measurementmethod depending on whether or not the area sensor mode is selected(step S47).

First, a description will be given in the case where the area sensormode is selected.

When the start-measurement switch is pressed, the X-axis motion stage422X and the Y-axis motion stage 422Y are moved to a measurementstarting position (S50). In this step, the computer PC retrieves themeasurement starting position which has been input and moves the X-axismotion stage 422X and the Y-axis motion stage 422Y to the measurementstarting position, and the cells CE are thus moved to a position withinthe imaging field of the objective lens 448.

Then, the shutter 435 is opened (step S51) and the objective lens 448 isselected (step S52). Here, the computer PC drives the revolver 447 toselect an objective lens 448 having a predetermined magnification on thebasis of the specified measurement magnification.

Next, the filter unit 445 is selected (step S53). Here, the filtercontrol unit 446C selects the excitation filter 456, the absorptionfilter 458, and so on that are most appropriate for the measurement onthe basis of the fluorescent protein specified in the computer PC.

The operations carried out from when the start-measurement switchdescribed above is pressed up to this point (steps S50 to S53) areautomatically selected and executed according to the measurement mode.

After that, the focus position is detected (step S54), and then imageacquisition is performed and the image data is output to an image memoryin the computer PC (step S55).

Then, if the required image acquisition has not yet been completed, theoperations from selection of the objective lens 448 (step S52) to imageacquisition and output of the image data to the image memory in thecomputer PC (step S55) are repeated until the required image acquisitionis completed (step S56).

When the required image acquisition has been completed, the X-axismotion stage 422X or the Y-axis motion stage 422Y is driven by one step(step S57). Then, if the position to which the X-axis motion stage 422Xor the Y-axis motion stage 422Y has been moved is within the measurementregion, the operations from selection of the objective lens 448 (stepS52) to driving the motion stage by one step (step S57) are repeated.These operations are repeated until the position to which the X-axismotion stage 422X or the Y-axis motion stage 422Y has been moved isoutside the measurement region (step S58).

When the position of the X-axis motion stage 422X or the Y-axis motionstage 422Y is outside the measurement region, the shutter 435 is closed(step S59).

Thereafter, once the predetermined measurement time interval is over,the operations from opening of the shutter 435 (step S51) to closing ofthe shutter 435 (step S59) are repeated until the end of the measurementtime (step S60).

Next, a case where the area sensor mode is not selected shall bedescribed.

When the start-measurement switch is pressed, the x-axis motion stage422X and the Y-axis motion stage 422Y are moved to the measurementstarting position (step S70). Here, the computer retrieves themeasurement start position that was input and moves the X-axis motionstage 422X and the Y-axis motion stage 422Y to the measurement startingposition to move the cells CE to a position within the imaging field ofthe objective lens 448.

Then, the shutter 435 is opened (step S71), and the focus position isdetected (step S72).

Next, the objective lens 448 is selected (step S73). Here, the computerPC drives the revolver 447 to select an objective lens 448 having apredetermined magnification, on the basis of the specified measurementmagnification.

Next, the filter unit 445 is selected (step S74). Here, the filtercontrol unit 446C selects the excitation filter 456, the absorptionfilter 458, and so on that are most appropriate for the measurement onthe basis of the fluorescent protein specified in the computer PC. Theoperations carried out from when the start-measurement switch is pressedup to this point (steps S70 to S74) are automatically selected andexecuted according to the measurement mode.

After that, driving of the X-axis motion stage 422X and the Y-axis stage422Y commences (step S75), and then image acquisition is performed andthe image data is output to an image memory in the computer PC (stepS76).

Then, if the required image acquisition has not yet been completed, theoperations from selection of the objective lens 448 (step S73) to imageacquisition and output of the image data to the memory in the computerPC (step S76) are repeated until the required image acquisition iscompleted (step S77).

When the required image acquisition has been completed, the shutter 435is closed (step S78).

Thereafter, once the predetermined measurement time interval is over,the operations from opening of the shutter 435 (step S71) to closing ofthe shutter 435 (step S78) are repeated until the end of the measurementtime (step S79).

When image acquisition of the cells CE has been completed, the acquiredimages are then processed. Therefore, the method of processing theacquired images will be explained with reference to FIG. 39.

FIG. 39 is a flowchart showing the image processing method.

First, the image processing unit of the computer PC extracts thebackground image from the acquired images collected in the memory (stepS91), and removes the background image from the acquired images (stepS92).

Next, the maximum brightness region of the image, which can be enhanced,is read out (step S93) and multiplied by, for example, a predeterminedcoefficient to enhance the image (step S94). With this processing, theimage is enhanced so that the individual cells CE can easily berecognized as spots in the image from which the background is removed.

Then, by extracting portions having a brightness higher than, forexample, a predetermined threshold from the enhanced image, the brightcells CE can be clearly recognized as individual spots (step S95).

Next, geometrical features, such as the center of gravity and the area,chemical features, and optical features, such as the fluorescenceintensity, of the cells CE are more accurately determined, andpositional information of the cells CE is determined and extracted (stepS96). Extracting these features allows the individual cells CE to bedistinguished.

After extracting the features of the cells CE, correction (step S97) ofthe enhancement (S94) carried out for recognizing the cells CE iscarried out. With this correction, the effect of the predeterminedcoefficient used for enhancing the image is removed.

Next, after correction, the features are output to a file, for example,and are stored in that file (step S98).

Accordingly, the image processing unit of the computer PC can convertthe fluorescence distribution of the cells CE at each position on theentire surface of the slide glass, microplate, or the like into animage. Also, since the image processing unit can accurately track theindividual cells CE, it can target a predetermined number of cells CE,which allows localized, long-term measurement of the fluorescencedistribution of the cells CE while performing culturing. Furthermore,while culturing the cells CE, the entire surface of the slide glass,microplate, or the like is measured at fixed time intervals, forexample, and the fluorescence intensity of the cells CE over time can beautomatically measured.

Next, data processing carried out after extracting data about thefeatures of the cells CE from the acquired images will be described withreference to FIG. 40.

FIG. 40 is a flowchart depicting the data processing procedure.

Here, processing of the cell data (features) stored in the file iscarried performed by a data processing unit of the computer PC.

First, the data processing unit reads out (step S101) raw data(features) of the cells CE, which is stored in the file, and sorts thedata to arrange it time-sequentially for each cell CE (step S102). Whenthe data has been sorted, the data processing unit graphs the variationin brightness of each cell CE, that is, the level of expression, withtime (step S103).

When the graphing has been completed, the data processing unit displaysa preview of the graph (step S104), and outputs the graph data to a file(step S105).

By performing this processing, when cells CE are cultured for anextended period of time, the variation of a single cell with time can beeasily examined. Therefore, during culturing, the variation in the levelof expression of the cells CE with time can be accurately and easilymeasured.

Next, adjustment of the irradiation intensity carried out duringmeasurement of the cells CE will be explained with reference to FIG. 41.

FIG. 41 is a flowchart showing the procedure for adjusting theintensity.

First, the intensity of light irradiated onto the cells CE is measured(step S111). The irradiation intensity may be calculated from the outputof the light-intensity monitor 450, by providing an irradiance meter formeasuring the intensity, or by providing a power meter and calculatingthe intensity from the output of the power meter.

If the measured irradiation intensity is within a permissible range, theprocess returns to measurement of the irradiation intensity (step S111)and repeats this until the irradiation intensity is outside thepermissible range (step S112).

Once the irradiation intensity is outside the permissible range, the NDfilter (not shown) included in the light-intensity adjusting mechanism443 is changed (step S113) to adjust the irradiation intensity so thatit falls within the permissible range. Thereafter, the process returnsto measurement of the irradiation intensity (step S111), and repeats theadjustment of the irradiation intensity.

Next, a control method for supplying and replacing the culture fluid inthe chamber 510 is described with reference to FIG. 42.

FIG. 42 is a flowchart showing the method for supplying and replacingthe culture fluid.

First, the background level of the acquired image is analyzed (stepS121). The autofluorescence of the culture fluid in the background ofthe acquired image is acquired, and the brightness of thisautofluorescence is analyzed.

Here, since the brightness of this autofluorescence increases as theculture fluid ages, the point at which the culture fluid should bereplaced can be detected by measuring the brightness of thisautofluorescence.

Then, if the temporal variation in the analyzed background level iswithin a predetermined level, the process returns to analysis of thebackground level (step S121) and repeats this until the temporalvariation of the background level exceeds the predetermined level (stepS122).

Once the temporal variation of the background level exceeds thepredetermined value, the waste pump 482 for the culture fluid isoperated (step S123), and then, the supply pump 481 for the culturefluid is operated (step S124).

The intervals at which the culture fluid should be supplied and replacedmay be determined on the basis of the autofluorescence of the culturefluid, as described above. Alternatively, the culture fluid may besupplied/replaced continuously, or automatically at intervals specifiedin advance by the user. Alternatively, the point at which the culturefluid should be replaced can be specified at will by selecting the typeof cells CE from a table that is registered in advance. In addition, theamount of culture fluid to be replaced may be specified by the user ormay be determined on the basis of the autofluorescence of the culturefluid. Alternatively, the amount of culture fluid to be replaced can bespecified at will by selecting the type of cells CE from a table that isregistered in advance.

The amount of culture fluid to be replaced may be calculated anddetermined automatically using the weight and so on.

In this embodiment, the level of autofluorescence in the background isdetected using the acquired image; however, it may be detected from anacquired image of a location where cells CE do not exist. Alternatively,it may be detected by providing an optical detector in the vicinity ofthe culture-fluid vessel 472.

According to the measurement procedure described above, as shown in FIG.43, a cell-tracking image showing the change in position of individualcells over time can be obtained.

Next, the culturing and measurement procedures when using the microplate520 will be described with reference to FIGS. 44 and 45.

FIGS. 44 and 45 are flowcharts showing the culturing and measurementwhen using the microplate 520.

First, sterilized water is supplied to the reservoir 521 in theincubator box 500 a (step S131).

Next, the PC is started up (step S132), and the main power supplies forthe detection unit 420 and the culture unit 470 are turned on (stepS133).

After that, the internal fan 522 inside the incubator box 500 a isoperated (step S134) to circulate the air inside the incubator box 500a. Then, the CO₂-concentration controller 495 is operated (step S135) toregulate the carbon dioxide concentration in the culture gas supplied tothe incubator box 500 a at 5%. Thereafter, the temperature controllersare operated (step S13.6) to regulate the culture-fluid temperature, theculture-gas temperature, and the temperature inside the insulatedcompartment 421 at about 37° C.

After that, the door 427 of the detection unit 420 is opened (stepS137), the incubator box 500 a is placed on the stage 422 (step S138),and the door 427 is closed (step S139).

Next, the transmission light source 423 is turned on (step S140) toirradiate the cells CE with transmission light, and the measurementconditions are set (step S141).

Then, by pressing the start-measurement button (step S142), measurementof the cells CE commences.

First, scanning is carried out beforehand on predetermined cells CE toperform autofocusing (step S143), and once the focus position of eachpart has been determined, the shutter 435 is opened (step S144).

Next, an image of the cells CE is acquired and output (step S145). Here,the acquired image data is output to the memory of the computer PC.

Then, if the required image acquisition has not yet been completed, theoperations from the autofocusing (step S143) to the image acquisitionand outputting (step S145) are repeated until the required imageacquisition has been completed (step S146). Here, the required imagesare, for example, the images acquired using the selected wavelength, theimages acquired using the selected magnification, and so forth.

Once the required image acquisition has been completed, the X-axismotion stage 422X or the Y-axis motion stage 422Y is driven by one step(step S147). Then, if the position to which the X-axis motion stage 422Xor the Y-axis motion stage 422Y has moved is within the measurementregion, the operations from the autofocusing (step S143) to driving ofthe motion stage by a single step (step S147) are repeated. Theseoperations are repeated until the position to which the X-axis motionstage 422X or the Y-axis motion stage 422Y has moved is outside of themeasurement region.

When the position to which the X-axis motion stage 422X or the Y-axismotion stage 422Y has moved is outside the measurement region, theshutter 435 is closed (step S149), and the X-axis motion stage 422X andthe Y-axis motion stage 422Y are moved to their home positions (stepS150).

Thereafter, after a predetermined measurement interval, the operationsfrom the autofocusing (step S143) to moving the stages to their homepositions (step S150) are repeated until the end of the measurement time(step S151).

Once the measurement time ends (step S152), the door 427 is opened (stepS153), and the microplate 520 is removed from the incubator box 500 a(step S154). Then, the sterilized water is removed from the reservoir521 (step S155), and the door 427 is closed (S156).

After that, the UV lamp 425 inside the insulated compartment 421 isilluminated (step S157) to sterilize the interior of the insulatedcompartment 421, and the measurement is thus completed.

As described above, the sterilization may be carried out at the end ofthe measurement procedure, or alternatively, it may be carried out atthe beginning of the measurement procedure to sterilize the insulatedcompartment 421 before measurement actually takes place.

The autofocusing may be carried out for each measurement of the cellsCE, as described above, but it need not be carried out for eachmeasurement.

With the configuration described above, since the thermal environment ismaintained by the insulated compartment 421 and the humidity environmentand the culture-fluid environment are maintained by the chamber 510disposed inside the insulated compartment 421, the humidity environmentand the culture-fluid environment are influenced by the thermalenvironment, and therefore the thermal environment inside the chamber510 is also maintained.

Accordingly, sudden changes and non-uniformities in the thermalenvironment, which might cause damage to the cells CE, are moderated viathe humidity environment and the culture-fluid environment, andtherefore, it is possible to reduce damage caused to the cells CE.

Also, since the dimensions of the chamber 510 are small compared to theinsulated compartment 421, it is easier to maintain and control thehumidity environment and the culture-fluid environment, thus making itrelatively difficult to cause damage to the cells CE.

Since the cells CE can be examined through the insulated compartment421, the incubator box 500, and the chamber 510, it is possible to carryout examination while culturing the cells CE, without causing any damageto the cells CE. Accordingly, behavior that the cells exhibit duringculturing can be accurately measured over time.

It is possible to measure in real time the reaction of the cells CE inthe object under examination while changing the culture conditions. Forexample, the existence and level of expression of proteins can bemeasured, and changes in the level of expression with time can beaccurately measured.

Furthermore, deterioration of the activity of the cells CE by handlingthem in a first examination can be prevented, which enables multipleexaminations of the same cells CE. Also, since multiple examinations ofthe same cells CE can be carried out at intervals, it is not necessaryto control the experimental protocol.

Since the detection section 440 examines the cells CE in the chamber 510via the insulated compartment 421 and the incubator 500, it is notnecessary to insert and remove the cells CE from the chamber 510 duringexamination, and therefore, the cells CE can remain inside the chamber510 during examination. Accordingly, it is possible to accuratelyexamine the same position in each examination. Also, contamination canbe prevented during examination, which can prevent the cells from beingstressed.

Furthermore, by controlling the environmental conditions inside thechamber 510 (for example, the combination of carbon dioxideconcentration and humidity), it is possible to prevent the performanceof the detection section 440 from deteriorating.

Moreover, since the cells CE are contained inside the chamber 510, whichis disposed inside the insulated compartment 421 and the incubator box500, a certain distance can be maintained between the cells CE and theenvironment outside the incubator box 500 compared to a case wherechamber 510 is not provided. Therefore, it is possible to reduce theeffects of electric and magnetic fields from outside the incubator box500, such as those from the driving motors of the X-axis motion stage422X and the Y-axis motions stage 422Y and magnets provided in the door427.

Eleventh Embodiment

Next, an eleventh embodiment will be described with reference to FIGS.46A to 48.

The basic construction of the biological-specimen examination system ofthis embodiment is the same as the tenth embodiment, but theconstructions of the detection unit and the culture unit are differentfrom those in the tenth embodiment. Therefore, in this embodiment, onlythe detection unit and the culture unit will be described using FIGS.46A to 48, and a description of the chamber and so on will be omitted.

FIG. 46A is an elevational view of a biological-specimen examinationsystem 600 of this embodiment, and FIG. 46B is a side view thereof.

As shown in FIGS. 46A and 46B, the biological-specimen examinationsystem 600 includes an inverted microscope (microscope imagingapparatus) 610 and a culture stage 620. The inverted microscope 610 andthe culture stage 620 may be integrated or they may be constructed so asto be detachable from each other.

If the culture system 620 can be attached to and detached from theinverted microscope 610, an existing inverted microscope can also beused. In such a case, even though a stage driving motor is disposed inthe vicinity of the cells CE due to constraints on the shape and theconstruction involved with attaching the culture stage 620, for example,the effect of electric and magnetic fields on the cells CE can besuppressed.

Also, when examining the cells CE using the inverted microscope 610, forexample, the culture stage 620 can be attached to the invertedmicroscope 610; at other times (for example, when culturing the cellsCE), the inverted microscope 610 can be removed from the microscope.

FIG. 47 is a plane view of the culture stage 620, and FIG. 48 is aperspective view of the culture stage 620.

As shown in FIGS. 46A, 46B, and 47, the culture stage 620 includes aframe 621, openable/closable lid 622 provided on the upper surface ofthe frame 621, an X-axis motion stage 422X, a Y-axis motion stage 422Y,a small or strip-shaped heater 620H, a heatsink 623, a fan 624, andculture-gas supply connector 625.

The frame 621 is preferably formed of a highly opaque,corrosion-resistant material, such as anodized aluminum or stainlesssteel, like SUS 316. More preferably, from the viewpoint of thermalinsulation, a material having a low thermal conductivity is selected.

The interior of the frame 621 is divided into a measurement area 626 forperforming examination of cultured cells and a non-measurement area 627only for culturing cells. Thus, cell culturing is performed in theculture stage 620. A microplate 520 that holds the cells is accommodatedinside the culture stage 620, and the culture stage 620 is configured soas to allow examination of the cells in the microplate 520 from outsidethe culture stage 620. In the description given here, the microplate 520is used as a culture vessel, as shown in FIGS. 47 and 48, but a dish orflask may also be used.

The fan 624 and the culture-gas supply connector 625 are disposed in aside wall in the non-measurement area 627 in the frame 621. Also, theheater 620H and the heatsink 623 for dissipating heat from the heater620H are disposed in an area where the fan 624 and the culture-gassupply connector 625 are not disposed (including the measurement area626).

The fan 624 causes convection of the air inside the culture stage 620and is arranged so as not to blow directly onto the incubator box 500 a.

The temperature inside the culture stage 620 is raised by means of theheater 620H and is regulated at 36.5° C.+0.5° C.

As shown in FIGS. 47 and 48, the X-axis motion stage 422× and the Y-axismotion stage 422Y are provided on the bottom surface of the frame 621.The X-axis motion stage 422× and the Y-axis motion stage 422Y are drivenby, for example, motors and ball screws.

A small or strip-shaped heater (not shown) is attached to the Y-axismotion stage 422Y. The heater is disposed at a position such that themicroplate 520 is uniformly heated.

The measurement area 626 and the non-measurement area 627 are formed bydividing the interior of the culture stage 620 in the X-direction bymeans of a top plate 628 and a pair of partition seats 629, which arefixed to the frame 621. The region to the left of the partition seats629 in FIG. 47 constitutes the measurement area 626, and the region tothe right constitutes the non-measurement area 627.

A partition plate 629 a that is formed in substantially the same shapeas the cross-sectional shape of the frame 621 is attached to the X-axismotion stage 422X.

By moving the X-axis motion stage 422X towards the non-measurement area627, side faces at both ends (the ends in the Y-direction) of thepartition plate 629 a come into contact with the partition seats 629,and the partition plate 629 a is thus positioned so as to divide thespace inside the frame 621 into two portions in the left-to-right(X-axis) direction.

Furthermore, since the edge of the partition plate 629 a at the topplate 628 is positioned in contact with the lower surface of the topplate 628, the measurement area 626 and the non-measurement area 627 candefine two spaces that are separated from each other.

At the upper opening of the measurement area 626, a glass lid 630 isremovably attached to the frame 621 and is disposed so as to cover theupper opening of the measurement area 626. The glass lid 630 can beattached by, for example, screwing the glass lid 630 to the frame 621,or by means of a lock mechanism, a hook, a magnet, and so forth.

The glass lid 630 may be constructed such that the entire surface orsubstantially the entire surface except for the peripheral frame portionis formed of the glass plate 631, or it may be formed with a minimumpossible area so long as it does not obstruct measurement. In order tosuppress the reflection of light during transmission examination andincidence examination, it is preferable to use an optical glass materialhaving an anti-reflection film (AR coat) coated on both sides thereof asthe glass plate 631.

The anti-reflection film may be coated on both sides of the glass plate631, as described above, or it may be coated on only one side of theglass plate 631.

The glass lid 630 may be removed as required when carrying out varioustasks, for example, when changing the objective lens of the invertedmicroscope 610, when cleaning the inside of the measurement area 626,and so forth.

The glass lid 630 may be provided with an observation hole into whichthe objective lens of the inverted microscope 610 is inserted. Also, arubber sheet may be disposed in the observation hole to occupy a gapbetween the objective lens and the observation hole. The sheet ispreferably disposed so as to prevent relative motion between theobjective lens and the culture stage 620.

At the upper opening of the non-measurement area 627, theopenable/closable lid 622 is attached so that it can be opened andclosed by means of a hinge or the like. When closed, one edge of theopenable/closable lid 622 is in contact with the top plate 628 so as tobe supported.

The openable/closable lid 622 is entirely formed of an opaque material(for example, the same material as the frame 621) and is provided withan observation-hole cover 632 that blocks the observation hole or aUV-irradiation-hole cover 633 that blocks a UV irradiation hole, asrequired.

The observation hole is an opening (window) formed in theopenable/closable lid 622. The observation-hole cover 632 is formed, forexample, of a glass plate, a resin plate, or the like having lowtransmittance and is inserted in the observation hole. Also, theobservation-hole cover 632 is formed of the same opaque material as theopenable/closable lid 622 and may be attached in such a manner that itcan be either detached or opened and closed.

The UV-irradiation-hole is an opening (window) formed in theopenable/closable lid 622. The UV-irradiation-hole cover 633 is formed,for example, of the same opaque material as the openable/closable lid622 and is attached in such a manner that it can be opened and closed,or detached from the UV irradiation hole.

The UV-irradiation-hole cover 633 is removed when irradiating theinterior of the non-measurement area 627 with ultraviolet (UV) light tosterilize it. A handheld UV lamp can be used for the UV irradiation.

The incubator box 500 a, which contains the microplate 520, is held onthe Y-axis motion stage 422Y. Since the incubator box 500 a is the sameas that described in the tenth embodiment, the same components areassigned the same reference numerals, and a description thereof shall beomitted.

Culture gas is supplied to the connector 523 of the incubator box 500 avia a culture-gas supply tube 634 from a culture-gas supply connector625 of the culture stage 620.

With the configuration described above, since the biological-specimenexamination system 600 according to the present invention includes theintegrated inverted microscope 610, a biological specimen can beexamined using the inverted microscope 610. Therefore, more detailedexamination can be carried out compared to a case where the invertedmicroscope 610 is not provided.

When measuring and culturing the cells, in order that the cells are notaffected by ambient light, the entire biological-specimen examinationsystem 600 may be surrounded with a blackout curtain.

Also, instead of cells, the biological specimen to be measured may bevarious other types of biological specimen, such as bacteria,microorganisms, ova, and so forth.

With the biological-specimen examination system of this embodiment,since it is possible to reduce the image acquisition time, specialphenomena that occur in the biological specimen only for a short timecan be observed, and the accuracy of the examination results of thebiological specimen can be improved.

Furthermore, with the biological-specimen examination system of thisembodiment, an advantage is afforded in that, even when the brightnessof the biological specimen changes over time, it is possible toeffectively observe such changes in the biological specimen over time.Also, it is possible to accurately examine a wide range of biologicalspecimens, from specimens having low brightness to specimens having highbrightness.

Moreover, with the biological-specimen examination system of thisembodiment, since the time required for measuring the measurement regiononce can be shortened, the motion of the biological specimen in part ofthe measurement region does not vary substantially over time. Thus, theaccuracy of the examination results when examining the biologicalspecimen over time can be improved.

1. A microscope imaging apparatus comprising: a stage that holds anobject under examination; an illumination unit that illuminates theobject under examination; an image-acquisition unit that acquires imagesof the object under examination; a motion unit that moves the stage andthe image-acquisition unit relative to each other; and a control unitthat controls the image-acquisition unit and the motion unit, whereinthe image-acquisition unit includes an imaging device that is capable ofacquiring images with the time delay integration method, and when imageacquisition of the object under examination is carried out a pluralityof times, the control unit makes the exposure times of the imagingdevice different for each image acquisition.
 2. A microscope imagingapparatus according to claim 1, wherein the control unit includes anexposure-time input unit for inputting the exposure time and anexposure-time setting unit for setting the length of the exposure timeto be used in the subsequent image acquisition, the exposure time to beused in the subsequent image acquisition being determined on the basisof the image obtained by image acquisition using the input exposuretime.
 3. A microscope imaging apparatus according to claim 2, whereinthe exposure time input to the exposure-time input unit defines amaximum exposure time serving as the upper limit for the exposure time.4. A microscope imaging apparatus according to claim 1, wherein theimaging device in the image-acquisition unit has an anti-smear function.5. A microscope imaging apparatus according to claim 1, wherein thecontrol unit combines images having different exposure times into asingle image by carrying out predetermined image processing.
 6. Amicroscope imaging apparatus comprising: a stage that holds an objectunder examination; an illumination unit that illuminates the objectunder examination; an image-acquisition unit that acquires images of theobject under examination; a motion unit that moves the stage and theimage-acquisition unit relative to each other; and a control unit thatcontrols the image-acquisition unit and the motion unit, wherein theimage-acquisition unit includes an imaging device that is capable ofacquiring images with a time delay integration method, and by performinga prescan of the object under examination to obtain a detected intensityof the object under examination, the control unit determines an exposuretime for image acquisition after the prescan on the basis of thedetected intensity.
 7. A microscope imaging apparatus according to claim6, wherein the illumination unit includes a first objective lens and asecond objective lens that focus light onto the object underexamination; during the prescan, the first objective lens is disposed ina light path of the illumination unit; during image acquisition afterthe prescan, the second objective lens is disposed in the light path ofthe illumination unit; and the magnification of the first objective lensis lower than the magnification of the second objective lens.
 8. Amicroscope imaging apparatus according to claim 6, comprising: avariable-power lens that can be inserted in and removed from the lightpath of the illumination unit.
 9. A microscope imaging apparatusaccording to claim 6, wherein the control unit includes: amaximum-exposure-time input unit; and a selection unit, wherein themaximum exposure time of the object under examination for imageacquisition after the prescan is input to the maximum-exposure timeinput unit, and using the selection unit, the maximum exposure time andthe exposure time determined in the prescan are compared, and on thebasis of this comparison, the shorter exposure time is set as theexposure time used for measurement of the object under examination. 10.A microscope imaging apparatus according to claim 6, wherein the objectunder examination has a plurality of sample located portions formedthereon, the individual sample located portions having substantially thesame brightness, and the image-acquisition unit acquires images of theplurality of sample located portions.
 11. A microscope imaging apparatuscomprising: a stage that holds an object under examination; anillumination unit that illuminates the object under examination; animage-acquisition unit that acquires images of the object underexamination; a motion unit that moves the stage and theimage-acquisition unit relative to each other; and a control unit thatcontrols the image-acquisition unit and the motion unit, wherein theimage-acquisition unit includes an imaging device that is capable ofimage acquisition using two methods; the control unit includes: anexamination-object-parameter input unit for inputting information aboutthe object under examination as an examination-object parameter; acalculation unit that calculates a time required for the relative motionon the basis of the examination-object parameter which has been input;and a switching unit that changes the image-acquisition method of theimaging device on the basis of the calculation result; and the twoimage-acquisition methods are a time delay integration method and atwo-dimensional imaging method in which accumulated charge is producedby a single exposure.
 12. A microscope imaging apparatus according toclaim 11, wherein the examination-object parameter is the wavelengthused in the image acquisition.
 13. A microscope imaging apparatusaccording to claim 11, wherein the examination-object parameter is theexposure time used in the image acquisition.
 14. A microscope imagingapparatus according to claim 11, wherein the examination-objectparameter is the density of the object under examination.
 15. Abiological-specimen examination system comprising: a culture unit forculturing a biological specimen; and a detection unit disposed adjacentto the culture unit, wherein the detection unit includes: a microscopeimaging apparatus according to claim 1; and a preserving unit forpreserving the biological specimen in a predetermined state.